Human Anti-NGF Neutralizing Antibodies as Selective NGF Pathway Inhibitors

ABSTRACT

This invention provides antibodies that interact with or bind to human nerve growth factor (NGF) and neutralize the function of NGF thereby. The invention also provides pharmaceutical compositions of said antibodies and methods for neutralizing NGF function, and particularly for treating NGF-related disorders (e.g., chronic pain) by administering a pharmaceutically effective amount of anti-NGF antibodies. Methods of detecting the amount of NGF in a sample using anti-NGF antibodies are also provided.

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 12/277,919, filed Nov. 25, 2008, which is acontinuation application of U.S. patent application Ser. No. 10/891,658,filed Jul. 15, 2004, and claims the benefit of priority to U.S.provisional application Ser. No. 60/487,431, filed Jul. 15, 2003. Thisapplication is also related to U.S. patent application Ser. No.11/767,326, filed Jun. 22, 2007, which is a divisional application ofU.S. Ser. No. 10/891,658. The disclosures of all these applications areincorporated by reference herein.

The sequence listing is filed with the application in electronic formatonly and is incorporated by reference herein. The sequence listing textfile “02-1240-F-CIP.SeqList.txt” was created on Nov. 25, 2008, and is79,116 bytes in size.

FIELD OF THE INVENTION

The invention relates to human monoclonal antibodies that bind nervegrowth factor (NGF). Compositions and methods for treating pain andpain-related disorders are also described.

BACKGROUND OF THE INVENTION

Every day, more than two million people in the United States areincapacitated by chronic pain (Jessell and Kelly, 1991, “Pain andAnalgesia” in PRINCIPLES OF NEURAL SCIENCE, 3^(rd) Ed., (Kandel,Schwartz, and Jessell, ed.), Elsevier, N.Y.). Unfortunately, currenttreatments for pain are only partially effective, and many of thesetreatments themselves cause debilitating or dangerous side effects. Forexample, although non-steroidal anti-inflammatory drugs (“NSAIDs”) suchas aspirin, ibuprofen, and indomethacin are moderately effective againstinflammatory pain, they are also renal toxins, and high doses tend tocause gastrointestinal irritation, ulceration, bleeding, and mentalconfusion. Patients treated with opioids also frequently experienceconfusion, and long-term opioid use is associated with tolerance anddependence. Local anesthetics such as lidocaine and mexiletinesimultaneously inhibit pain and cause loss of normal sensation.

Pain is a perception based on signals received from the environment andtransmitted and interpreted by the nervous system (for review, seeMillan, 1999, Prog. Neurobiol. 57:1-164). Noxious stimuli such as heatand touch cause specialized sensory receptors in the skin to sendsignals to the central nervous system (“CNS”). This process is callednociception, and the peripheral sensory neurons that mediate it arenociceptors. Depending on the strength of the signal from thenociceptor(s) and the abstraction and elaboration of that signal by theCNS, a person may or may not experience a noxious stimulus as painful.When one's perception of pain is properly calibrated to the intensity ofthe stimulus, pain serves its intended protective function. However,certain types of tissue damage cause a phenomenon, known as hyperalgesiaor pronociception, in which relatively innocuous stimuli are perceivedas intensely painful because the person's pain thresholds have beenlowered. Both inflammation and nerve damage can induce hyperalgesia.Persons afflicted with inflammatory conditions, such as sunburn,osteoarthritis, colitis, carditis, dermatitis, myositis, neuritis,collagen vascular diseases (which include rheumatoid arthritis andlupus) and the like, often experience enhanced sensations of pain.Similarly, trauma, surgery, amputation, abscess, causalgia, collagenvascular diseases, demyelinating diseases, trigeminal neuralgia, cancer,chronic alcoholism, stroke, thalamic pain syndrome, diabetes, herpesinfections, acquired immune deficiency syndrome (“AIDS”), toxins andchemotherapy cause nerve injuries that result in excessive pain.

As the mechanisms by which nociceptors transduce external signals undernormal and hyperalgesic conditions become better understood, processesimplicated in hyperalgesia can be targeted to inhibit the lowering ofthe pain threshold and thereby lessen the amount of pain experienced.

Neurotrophic factors have been shown to play significant roles in thetransmission of physiologic and pathologic pain. Nerve growth factor(NGF) appears to be particularly important (for review, see McMahon,1996, Phil. Trans. R. Soc. Lond. 351:431-40; and Apfel, 2000, TheClinical Journal of Pain 16:S7-S11). Both local and systemicadministration of NGF have been shown to elicit hyperalgesia andallodynia (Lewin et al., 1994, Eur. J. Neurosci. 6:1903-1912).Intravenous infusion of NGF in humans produces a whole body myalgiawhile local administration evokes injection site hyperalgesia andallodynia in addition to the systemic effects (Apfel et al., 1998,Neurology 51:695-702). There is also a considerable body of evidenceimplicating endogenous NGF in conditions in which pain is a prominentfeature. For example, NGF is upregulated in dorsal root ganglion (DRG)Schwann cells for at least 2 months following peripheral nerve injuryand increased levels have been reported in the joints of animalssuffering from a variety of arthritis models (e.g., Aloe et al., 1993,Growth Factors 9:149-155). In humans, NGF levels are elevated insynovial fluid from patients with rheumatoid or other types of arthritis(e.g., Aloe et al., 1992, Arthritis and Rheumatism 35:351-355).Furthermore, it has been demonstrated that antagonism of NGF functionprevents hyperalgesia and allodynia in models of neuropathic and chronicinflammatory pain. For example, in animal models of neuropathic pain(e.g. nerve trunk or spinal nerve ligation) systemic injection ofneutralizing antibodies to NGF prevents both allodynia and hyperalgesia(Ramer et al., 1999, Eur. J. Neurosci. 11:837-846; and Ro et al., 1999,Pain 79:265-274). Examples of anti-NGF antibodies known in the artinclude, for example, PCT Publication Nos. WO 01/78698, WO 01/64247, WO02/096458, and WO 2004/032870; U.S. Pat. Nos. 5,844,092, 5,877,016, and6,153,189; Hongo et al., 2000, Hybridoma 19:215-227; Hongo et al., 1993,Cell. Mol. Biol. 13:559-568; and GenBank Accession Nos. U39608, U39609,L17078, or L17077.

Clearly, there is a need for new safe and effective treatments for pain,particularly by targeting small molecule mediators or exacerbators ofpain such as NGF.

SUMMARY OF THE INVENTION

This invention provides novel human monoclonal antibodies that aretherapeutically useful for managing pain. Specifically, the inventionprovides monoclonal antibodies that bind to nerve growth factor (NGF).Preferably, the monoclonal antibodies are human monoclonal antibodiesand neutralize biological activities of NGF and are useful forameliorating the effects of NGF-mediated pain responses. Also providedby the invention are cells that produce, and most preferably, secreteinto cell culture media the monoclonal antibodies of the invention. Inaddition to their use for treating and managing pain, the antibodies ofthe invention are useful for treating neuropathic and inflammatorypain-related responses.

The invention further provides fusion proteins comprising the sequenceof an antibody Fc region and one or more sequences identified as SEQ IDNO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQID NO: 20, SEQ ID NO: 22, and SEQ ID NOs: 79-130. Such molecules can beprepared using methods as described, for example, in InternationalPatent Application, Publication No. WO 00/24782, which is incorporatedby reference. Such molecules can be expressed, for example, in mammaliancells (e.g. Chinese Hamster Ovary cells) or bacterial cells (e.g. E.coli cells).

In certain aspects, the invention provides antibodies, preferablymonoclonal antibodies, most preferably human antibodies and humanmonoclonal antibodies, comprising a heavy chain and a light chain,wherein the heavy chain comprises an amino acid sequence as set forth inSEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof and thevariable region of the heavy chain comprises an amino acid sequence asset forth in SEQ ID NO: 10, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof. Preferably, the heavy chaincomprises an amino acid sequence as set forth in SEQ ID NO: 4.

In certain aspects, the invention provides antibodies, preferably humanantibodies, and more preferably monoclonal antibodies, most preferablyhuman monoclonal antibodies, comprising a heavy chain and a light chain,wherein the heavy chain comprises an heavy chain constant regionselected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgAand IgE heavy chain constant regions or any allelic variation thereof(as discussed in Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242), included herein byreference, and the variable region of the heavy chain comprises an aminoacid sequence as set forth in SEQ ID NO: 10, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof. Preferably,an antibody of the invention comprises an amino acid sequence of theIgG2 heavy chain constant region as set forth in SEQ ID NO: 4 or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof.

In certain aspects, the invention provides antibodies, preferably humanantibodies, and more preferably monoclonal antibodies, most preferablyhuman monoclonal antibodies, comprising a heavy chain and a light chain,wherein the light chain comprises an amino acid sequence as set forth inSEQ ID NO: 8 or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof and the light chain variable regioncomprises an amino acid sequence as set forth in SEQ ID NO: 12, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof.

In certain aspects, antibodies of the invention comprise a heavy chainand a light chain, wherein the variable region of the heavy chaincomprises an amino acid sequence as set forth in SEQ ID NO: 10, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof. In other aspects, the light chain variable region comprises anamino acid sequence as set forth in SEQ ID NO: 12, or an antigen-bindingor an immunologically functional immunoglobulin fragment thereof. Inadditional aspects, the heavy chain comprises an amino acid sequence asset forth in any of SEQ ID NO: 14, SEQ ID NO: 18, or SEQ ID NO: 20, oran antigen-binding or an immunologically functional immunoglobulinfragment thereof. In still further aspects, the light chain comprises anamino acid sequence as set forth in any of SEQ ID NO: 16, 20, 24, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof.

The invention also provides antibodies that bind specifically to NGF,wherein the heavy chain comprises a variable region comprising an aminoacid sequence as set forth in SEQ ID NO: 10, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and thelight chain comprises a variable region comprising an amino acidsequence as set forth in SEQ ID NO: 12, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof.

The invention further provides isolated human antibodies that bindspecifically to NGF, wherein the antibodies comprise:

(a) a heavy chain having a heavy chain variable region comprising anamino acid sequence as set forth in SEQ ID NO: 79, or an antigen-bindingor an immunologically functional immunoglobulin fragment thereof, and alight chain having a light chain variable region comprising an aminoacid sequence as set forth in SEQ ID NO: 80, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, (b) a heavychain having a heavy chain variable region comprising an amino acidsequence as set forth in SEQ ID NO: 81, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain having a light chain variable region comprising an amino acidsequence as set forth in SEQ ID NO: 82, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof,

(c) a heavy chain having a heavy chain variable region comprising anamino acid sequence as set forth in SEQ ID NO: 83, or an antigen-bindingor an immunologically functional immunoglobulin fragment thereof, and alight chain having a light chain variable region comprising an aminoacid sequence as set forth in SEQ ID NO: 84, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, or

(d) a heavy chain having a heavy chain variable region comprising anamino acid sequence as set forth in SEQ ID NO: 86, or an antigen-bindingor an immunologically functional immunoglobulin fragment thereof, and alight chain having a light chain variable region comprising an aminoacid sequence as set forth in SEQ ID NO: 87, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof.

In certain aspects, the invention also provides antibodies, comprising aheavy chain and a light chain, wherein the heavy chain comprises a heavychain variable region, and wherein the heavy chain variable regioncomprises a sequence that has at least 75%, preferably 80%, morepreferably at least 85%, even more preferably at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, and most preferably about 99%, identity tothe amino acid sequence as set forth in SEQ ID NO: 10, and wherein thelight chain comprises a light chain variable region, and wherein thelight chain variable region comprises a sequence that has at least 80%,preferably at least 85%, more preferably at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, and most preferably about 99%, identity to theamino acid sequence as set forth in SEQ ID NO: 12, wherein the antibodybinds specifically to NGF.

The invention also provides antibodies that bind specifically to NGF,wherein the heavy chain comprises an amino acid sequence as set forth inSEQ ID NO: 14 or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof, and the light chain comprises an aminoacid sequence as set forth in SEQ ID NO: 16, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof.

In certain aspects, the invention provides antibodies, comprising aheavy chain and a light chain, wherein the heavy chain comprises a heavychain variable region, and wherein the heavy chain variable regioncomprises a sequence that has at least 75%, preferably 80%, morepreferably at least 85%, even more preferably at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, and most preferably about 99%, identity tothe amino acid sequence as set forth in any of SEQ ID NO: 14, SEQ ID NO:18, or SEQ ID NO: 22, and wherein the light chain comprises a lightchain variable region, and wherein the light chain variable regioncomprises an amino acid sequence that has as least 80%, preferably atleast 85%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, and most preferably about 99%, identity to the amino acidsequence as set forth in SEQ ID NO: 16, wherein the antibody bindsspecifically to NGF.

The invention also provides single chain antibodies, single chain Fvantibodies, F(ab) antibodies, F(ab)′ antibodies and (Fab′)₂ antibodies.

In particular aspects, the invention provides a light chain comprisingan amino acid sequence as set forth in SEQ ID NO: 16, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof.

In addition, the invention provides a heavy chain comprising an aminoacid sequence as set forth in any of SEQ ID NO: 14, SEQ ID NO: 18, orSEQ ID NO: 22, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof.

The invention also relates to isolated human antibodies thatspecifically bind NGF, wherein the antibody comprises: (a) human heavychain framework regions, a human heavy chain CDR1 region, a human heavychain CDR2 region, and a human heavy chain CDR3 region; and (b) humanlight chain framework regions, a human light chain CDR1 region, a humanlight chain CDR2 region, and a human light chain CDR3 region. In certainaspects, the human heavy chain CDR1 region can be the heavy chain CDR1region of the monoclonal antibody (mAb) designated 4D4 as shown in SEQID NO:22 and the human light chain CDR1 region can be the light chainCDR1 region of mAb 4D4 as shown in SEQ ID NO:24. In other aspects, thehuman heavy chain CDR2 region can be the heavy chain CDR2 region of mAb4D4 as shown in SEQ ID NO:18 and the human light chain CDR2 region canbe the light chain CDR2 region of mAb 4D4 as shown in SEQ ID NO:20. Instill other aspects, the human heavy chain CDR3 region is the heavychain CDR3 region of mAb 4D4 as shown in SEQ ID NO:14, and the humanlight chain CDR3 region is the light chain CDR3 region of mAb 4D4 asshown in SEQ ID NO:16.

The invention also provides isolated human antibodies that specificallybind nerve growth factor, comprising a heavy chain and a light chain,wherein the heavy chain comprises a heavy chain variable regioncomprising an amino acid sequence as set forth in SEQ ID NO: 10, SEQ IDNO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, or SEQ ID NO: 87,or an antigen-binding or immunologically functional immunoglobulinfragments thereof.

The invention further provides isolated human antibodies thatspecifically bind NGF, comprising a heavy chain and a light chain,wherein the light chain comprises a light chain variable regioncomprising an amino acid sequence as set forth in SEQ ID NO: 12, SEQ IDNO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, or SEQ ID NO: 131, orantigen-binding or an immunologically functional immunoglobulinfragments thereof.

The antibodies of the invention are characterized by the capacity toantagonize at least one in vitro and/or in vivo activity associated withNGF polypeptides. Preferably, the invention provides isolated anti-humanNGF human antibodies with high affinity binding to NGF polypeptides,wherein the antibodies bind to a human NGF polypeptide and dissociatesfrom the human NGF polypeptide with a dissociation constant (K_(D)) ofabout 50×10⁻¹² M or less, as determined using KinExA, or which inhibitNGF induced survival in an in vitro neutralization assay with an IC₅₀ ofabout 1×10⁻⁸ M or less.

In a preferred embodiment, the invention provides an isolated anti-humanNGF human antibody that has the following characteristics:

a) inhibits NGF induced survival in an in vitro neutralization assaywith an IC₅₀ of about 1×10⁻⁹ M or less;

b) has a heavy chain CDR3 comprising the amino acid sequence of SEQ IDNO:14; and

c) has a light chain CDR3 comprising the amino acid sequence of SEQ IDNO:16.

The invention also provides isolated human antibodies or anantigen-binding or immunologically functional immunoglobulin fragmentsthereof that bind specifically to NGF with high affinity, wherein saidantibodies or fragments dissociate from a human NGF polypeptide with aK_(D) of about 1×10⁻⁹ or less and neutralizes human NGF bioactivity in astandard in vitro assay with an IC₅₀ of about 1×10⁻⁸ M or less, andwherein the antibodies or fragments comprise a heavy chain variableregion comprising:

a) a CDR1 region comprising an amino acid sequence of the formula:

a¹a²a³a⁴a⁵

wherein:

a¹ is a polar hydrophilic amino acid residue; a² is an aromatic aminoacid residue; a³ is a aliphatic, polar hydrophobic, aromatic amino acidresidue; a⁴ is a neutral hydrophobic or aliphatic amino acid residue;and a⁵ is a aliphatic or polar hydrophilic amino acid residue;

b) a CDR2 region comprising an amino acid sequence of the formula:

b^(l)b²b³b⁴b⁵b⁶b⁷b⁸b⁹b¹⁰b¹¹b¹²b¹³b¹⁴b¹⁵b¹⁶b¹⁷

wherein:

b¹ is a aliphatic, polar hydrophobic, or aromatic amino acid residue; b²is an aliphatic hydrophobic amino acid residue; b³ is a polarhydrophilic or aromatic amino acid residue; b⁴ is a polar hydrophilic,hydrophobic, or aromatic amino acid residue; b⁵-b⁹ are independentlypolar hydrophilic or aliphatic amino acid residues; b¹⁰ is a polarhydrophilic, aromatic, or aliphatic amino acid residue; b¹¹ is anaromatic or hydrophobic amino acid residue; b¹² is an aliphatichydrophobic or polar hydrophilic amino acid residue; b¹³ is analiphatic, hydrophobic or polar hydrophilic amino acid residue; b¹⁴ andb¹⁶ are independently polar hydrophilic amino acid residues; b¹⁵ is analiphatic or aromatic hydrophobic amino acid residue; and b¹⁷ is analiphatic acidic amino acid residue; and

c) a CDR3 region comprising an amino acid sequence of the formula:

c¹c²c³c⁴c⁵c⁶c⁷c⁸c⁹c¹⁰c¹¹c¹²c¹³c¹⁴c¹⁵c¹⁶c¹⁷

wherein:

c¹ is absent or an aliphatic amino acid residue; c² is absent or a polarhydrophilic or an aromatic hydrophobic amino acid residue; c³ and c⁴ areindependently absent or a polar hydrophilic, aromatic hydrophobic, oraliphatic amino acid residues; c⁵ is absent or a polar hydrophilic,aliphatic or an aromatic amino acid residue; c⁶ is absent or a polarhydrophilic or aliphatic amino acid residue; c⁷is a polar hydrophilic oran aliphatic amino acid residue; c⁸ is a polar hydrophilic, hydrophobicor an aromatic amino acid residue; c⁹ is a polar hydrophilic, aliphaticor an aromatic hydrophobic amino acid residue; c¹⁰ polar hydrophilic,aromatic or an a liphatic hydrophobic amino acid residue; c¹¹-c¹³ areindependently polar hydrophilic or aromatic hydrophobic amino acidresidues; c¹⁴ is an aliphatic or aromatic hydrophobic amino acidresidue; c¹⁵ is a polar hydrophilic or neutral hydrophobic amino acidresidue; c¹⁶ is absent or a polar hydrophilic amino acid residue; andc¹⁷ is an aromatic hydrophobic or aliphatic hydrophobic amino acidresidue.

In one aspect, a¹ is a polar hydrophilic amino acid residue; a² is anaromatic hydrophobic amino acid residue; a³is an aliphatic hydrophobicamino acid residue; a⁴ is a neutral hydrophobic; a⁵ is a polarhydrophilic amino acid residue; b¹ is a aliphatic or aromatic amino acidresidue; b² is Ile; b³ is a polar hydrophilic amino acid residue; b⁴ isa polar hydrophilic or aromatic amino acid residue; b⁵-b⁹ areindependently polar hydrophilic or aliphatic amino acid residues; b¹⁰ isan aliphatic amino acid residue; b¹¹ is Tyr; b¹² is an aliphatichydrophobic amino acid residue; b¹³ is an aliphatic or polar hydrophilicamino acid residue; b¹⁴ and b¹⁶ are independently polar hydrophilicamino acid residues; and b¹⁵ is an aliphatic hydrophobic amino acidresidue; b¹⁷ is an aliphatic acidic amino acid residue; c¹ is absent oran aliphatic amino acid residue; c² is absent or a polar hydrophilic oran aromatic hydrophobic amino acid residue; c³ and c⁴ are independentlyabsent or a polar hydrophilic, aromatic hydrophobic, or aliphatic aminoacid residues; c⁵ is absent or a polar hydrophilic amino acid residue;c⁶ is absent or a polar hydrophilic or aliphatic amino acid residue; c⁷is a polar hydrophilic or an aliphatic amino acid residue; c⁸ is a polarhydrophilic, hydrophobic or an aromatic amino acid residue; c⁹ is apolar hydrophilic, aliphatic or an aromatic hydrophobic amino acidresidue; c¹⁰ is a polar hydrophilic, aromatic or an a liphatichydrophobic amino acid residue; c¹¹-c¹³ are independently polarhydrophilic or aromatic hydrophobic amino acid residues; c¹⁴ is analiphatic or aromatic hydrophobic amino acid residue; c¹⁵ is a polarhydrophilic or neutral hydrophobic amino acid residue; c¹⁶ is absent ora polar hydrophilic amino acid residue; and c¹⁷ is an aromatichydrophobic or aliphatic hydrophobic amino acid residue.

In a particular aspect, a¹ is Ser, Asp, or Thr; a² is Tyr; a³ is Ala,Ser, Trp, or Gly; a⁴ is Met or Ile; a⁵ is His, Gly, or Asn; b¹ is Tyr,Gly, Ile, or Asp; b² is Ile; b³ is Ser, Thr, Tyr, or Asn; b⁴ is Trp,Arg, or Pro; b⁵ is Ser, Asn, or Gly; b⁶ is Ser, Arg, Asp, or Gly; b⁷ isSer, His, or Gly; b⁸ is Ser, Ile, Asp, or Thr; b⁹ is Leu, Ile, or Thr;b¹⁰ is Gly, Lys, or Phe; b¹¹ is Tyr; b¹² is Ala or Ser; b¹³ is Asp, Gly,or Pro; b¹⁴ is Ser; b¹⁵ is Val or Phe; b¹⁶ is Lys or Gln; b¹⁷ is Gly; c¹is absent or an aliphatic amino acid residue; c² is absent or Tyr; c³and c⁴ are independently absent, Tyr, Asn, Val, or Glu; c⁵ is absent,Ser, Gly, or Trp; c⁶ is absent, Ser, Gly, Glu, or Leu; c⁷ is Gly, Arg,or Asp; c⁸ is Trp, Pro, Ser, or Thr; c⁹ is His, Gly, or Tyr; c¹⁰ is Val,Tyr, or Arg; c¹¹-c¹³ are independently Ser, Phe, Tyr, Asp, or Asn; c¹⁴is Phe, Val, or Gly; c¹⁵ is Met or Asp; c¹⁶ is absent, Asp, or Asn; andc¹⁷ is Tyr or Val.

In another particular aspect, a¹ is Ser or Asp; a² is Tyr; a³ is Ala orSer; a⁴ is Met or Ile; a⁵ is His or Asn; b¹ is Tyr or Gly; b² is Ile; b³is Ser, Thr, Tyr, or Asn; b⁴ is Trp, Arg, or Pro; b⁵ is Ser or Asn; b⁶is Ser or Arg; b⁷ is His or Gly; b⁸ is Ile or Thr; b⁹ is Leu, Ile, orThr; b¹⁰ is Gly or Phe; b¹¹ is Tyr; b¹² is Ala or Ser; b¹³ is Asp orGly; b¹⁴ is Ser; b¹⁵ is Val or Phe; b¹⁶ is Lys or Gln; b¹⁷ is Gly; c¹ isabsent or Gly; c² is absent or Tyr; c³ and c⁴ are independently absent,Tyr, Gly, or Val; c⁵ is absent or Ser; c⁶ is Ser or Gly; c⁷ is Gly orArg; c⁸ is Trp or Pro; c⁹ is His, Gly, or Tyr; c¹⁰ is Val or Tyr;c¹¹-c¹³ are independently Ser, Tyr, Phe, or Asp; c¹⁴ is Phe or Val; c¹⁵is Met or Asp; c¹⁶ is absent or Asp; and c¹⁷ is Tyr or Val.

-   -   In other particular aspects:    -   a) the heavy chain CDR1 has an amino acid sequence as set forth        in SEQ ID NO: 22, the heavy chain CDR2 has an amino acid        sequence as set forth in SEQ ID NO: 18, and the heavy chain CDR        3 has an amino acid sequence as set forth in SEQ ID NO: 14;    -   b) the heavy chain CDR1 has an amino acid sequence as set forth        in SEQ ID NO: 92, the heavy chain CDR2 has an amino acid        sequence as set forth in SEQ ID NO: 93, and the heavy chain CDR        3 has an amino acid sequence as set forth in SEQ ID NO: 94;    -   c) the heavy chain CDR1 has an amino acid sequence as set forth        in SEQ ID NO: 98, the heavy chain CDR2 has an amino acid        sequence as set forth in SEQ ID NO: 99, and the heavy chain CDR        3 has an amino acid sequence as set forth in SEQ ID NO: 100;    -   d) the heavy chain CDR1 has an amino acid sequence as set forth        in SEQ ID NO: 104, the heavy chain CDR2 has an amino acid        sequence as set forth in SEQ ID NO: 105, and the heavy chain CDR        3 has an amino acid sequence as set forth in SEQ ID NO: 106;    -   e) the heavy chain CDR1 has an amino acid sequence as set forth        in SEQ ID NO: 110, the heavy chain CDR2 has an amino acid        sequence as set forth in SEQ ID NO: 111, and the heavy chain CDR        3 has an amino acid sequence as set forth in SEQ ID NO: 112; and    -   f) the heavy chain CDR1 has an amino acid sequence as set forth        in SEQ ID NO: 116, the heavy chain CDR2 has an amino acid        sequence as set forth in SEQ ID NO: 117, and the heavy chain CDR        3 has an amino acid sequence as set forth in SEQ ID NO: 118.

The invention also provides an isolated human antibody or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof that binds specifically to NGF, wherein the antibody or fragmentcomprises a light chain variable region comprising:

a) a CDR1 region comprising an amino acid sequence of the formula:

a¹a²a³a⁴a⁵a⁶a⁷a⁸a⁹a¹⁰a¹¹a¹²

wherein:

a¹ is a polar hydrophilic amino acid residue; a², a¹¹ and a¹² areindependently aliphatic or hydrophobic amino acid residues; a³, a⁵, a⁷and a⁸ are independently aliphatic, polar hydrophilic, or hydrophobicamino acid residues; a⁴ is a polar hydrophilic amino acid residue; a⁶ isan aliphatic or hydrophobic amino acid residue; a⁹ is absent, or analiphatic or polar hydrophilic amino acid residue; and a¹⁰ is analiphatic, aromatic, or hydrophobic amino acid residue;

b) a CDR2 region comprising an amino acid sequence of the formula:

b¹b²b³b⁴b⁵b⁶b⁷

wherein:

b¹ is a aliphatic, polar hydrophobic, or hydrophobic amino acid residue;b² is an aliphatic or hydrophobic amino acid residue; b³ and b⁴ areindependently polar hydrophilic, aliphatic or hydrophobic amino acidresidues; b⁵ is a polar hydrophilic or aliphatic hydrophobic amino acidresidues; b⁶ is a polar hydrophilic or aliphatic hydrophobic amino acidresidue; and b⁷ is a polar hydrophilic amino acid residue; and

c) a CDR3 region comprising an amino acid sequence of the formula:

c¹c²c³c⁴c⁵c⁶c⁷c⁸c⁹c¹⁰c¹¹c¹²c¹³c¹⁴c¹⁵c¹⁶c¹⁷

wherein:

c¹ and c² are independently polar hydrophilic amino acid residues; c³ isa polar hydrophilic, aliphatic or hydrophobic amino acid residue; c⁴, c⁵and c⁶ are independently aliphatic, polar hydrophilic, or hydrophobicamino acid residues; c⁷ is absent or a polar hydrophilic or an aliphatichydrophobic amino acid residue; c⁸ is a polar hydrophilic or hydrophobicamino acid residue; and c⁹ is a polar hydrophilic amino acid residue,and wherein said antibody or fragment dissociates from a human NGFpolypeptide with a K_(D) of about 1×10⁻⁹ or less and neutralizes humanNGF bioactivity in a standard in vitro assay with an IC₅₀ of about1×10⁻⁸ M or less.

In one aspect, a¹, a³, a⁴, a⁷ and a⁸ are independently polar hydrophilicamino acid residues; a², a⁶, a¹¹ and a¹² are independently aliphatichydrophobic amino acid residues; a⁵ is a polar hydrophilic or aliphaticamino acid residue; a⁹ is absent, or an aliphatic or polar hydrophilicamino acid residue; a¹⁰ is an aliphatic or aromatic amino acid residue;b¹ is a aliphatic, polar hydrophobic, or hydrophobic amino acid residue;b² is an aliphatic hydrophobic amino acid residue; b³, b⁴ and b⁷ areindependently polar hydrophilic amino acid residues; b⁵ and b⁶ areindependently polar hydrophilic or aliphatic hydrophobic amino acidresidues; c¹ and c² are independently polar hydrophilic amino acidresidues; c³ is a polar hydrophilic, aliphatic or hydrophobic amino acidresidue; c⁴, c⁵, and c⁶ are independently aliphatic, polar hydrophilic,or hydrophobic amino acid residues; c⁷ is absent or an aliphatichydrophobic amino acid residue; c⁸ is a hydrophobic amino acid residue;and c⁹ is a polar hydrophilic amino acid residue.

In a particular aspect, a¹, a³, a⁴, and a⁷ are Arg, Ser, Gln, and Ser,respectively; a² is Ala; a⁵ is Gly or Ser; a⁸ is Ser or Ile; a⁹ isabsent, Ser, or Gly; a¹⁰ is Ala, Tyr, Trp or Phe; b¹ is Asp, Gly, Ala,or Val; b² and b³ are Ala and Ser, respectively; b⁴ is Ser or Asn; b⁵ isLeu or Arg; b⁶ is Glu, Ala, or Gln; b⁷ is Ser or Thr; c¹ and c² are Gln;c³ is Phe, Tyr, Arg, or Ala; c⁴ is Asn, Gly, or Ser; c⁵ is Ser or Asn;c⁶ is Tyr, Ser, Trp, or Phe; c⁷ is absent, Pro, or His; c⁸ is Leu, Trp,Tyr, or Arg; and c⁹ is Thr.

In another particular aspect, a¹, a², a³, a⁴, and a⁷ are Arg, Ala, Ser,Gln, and Ser, respectively; a⁵ is Gly or Ser; a⁸ is Ser or Ile; a⁹ isabsent, Ser, or Gly; a¹⁰ is Ala or Tyr; b¹ is Asp or Gly; b² and b³ areAla and Ser, respectively; b⁴ is Ser or Asn; b⁵ is Leu or Arg; b⁶ isGlu, Ala, or Gln; b⁷ is Ser or Thr; c¹ and c² are Gln; c³ is Phe, Tyr,Arg, or Ala; c⁴ is Asn, Gly, or Ser; c⁵ is Ser or Asn; c⁶ is Tyr, Ser,Trp, or Phe; c⁷ is absent, Pro, or His; c⁸ is Leu, Trp, Tyr, or Arg; andc⁹ is Thr.

-   -   In other particular aspects:    -   a) the light chain CDR1 has an amino acid sequence as set forth        in SEQ ID NO: 24, the light chain CDR2 has an amino acid        sequence as set forth in SEQ ID NO: 20, and the light chain CDR        3 has an amino acid sequence as set forth in SEQ ID NO: 16;    -   b) the light chain CDR1 has an amino acid sequence as set forth        in SEQ ID NO: 95, the light chain CDR2 has an amino acid        sequence as set forth in SEQ ID NO: 96, and the light chain CDR        3 has an amino acid sequence as set forth in SEQ ID NO: 97;    -   c) the light chain CDR1 has an amino acid sequence as set forth        in SEQ ID NO: 101, the light chain CDR2 has an amino acid        sequence as set forth in SEQ ID NO: 102, and the light chain CDR        3 has an amino acid sequence as set forth in SEQ ID NO: 103;    -   d) the light chain CDR1 has an amino acid sequence as set forth        in SEQ ID NO: 107, the light chain CDR2 has an amino acid        sequence as set forth in SEQ ID NO: 108, and the light chain CDR        3 has an amino acid sequence as set forth in SEQ ID NO: 109;    -   e) the light chain CDR1 has an amino acid sequence as set forth        in SEQ ID NO: 113, the light chain CDR2 has an amino acid        sequence as set forth in SEQ ID NO: 114, and the light chain CDR        3 has an amino acid sequence as set forth in SEQ ID NO: 115;    -   f) the light chain CDR1 has an amino acid sequence as set forth        in SEQ ID NO: 119, the light chain CDR2 has an amino acid        sequence as set forth in SEQ ID NO: 120, and the light chain CDR        3 has an amino acid sequence as set forth in SEQ ID NO: 121;    -   g) the light chain CDR1 has an amino acid sequence as set forth        in SEQ ID NO: 122, the light chain CDR2 has an amino acid        sequence as set forth in SEQ ID NO: 123, and the light chain CDR        3 has an amino acid sequence as set forth in SEQ ID NO: 124;    -   h) the light chain CDR1 has an amino acid sequence as set forth        in SEQ ID NO: 125, the light chain CDR2 has an amino acid        sequence as set forth in SEQ ID NO: 126, and the light chain CDR        3 has an amino acid sequence as set forth in SEQ ID NO: 127;    -   i) the light chain CDR1 has an amino acid sequence as set forth        in SEQ ID NO: 128, the light chain CDR2 has an amino acid        sequence as set forth in SEQ ID NO: 129, and the light chain CDR        3 has an amino acid sequence as set forth in SEQ ID NO: 130; and    -   j) the light chain CDR1 has an amino acid sequence as set forth        in SEQ ID NO: 132, the light chain CDR2 has an amino acid        sequence as set forth in SEQ ID NO: 133, and the light chain CDR        3 has an amino acid sequence as set forth in SEQ ID NO: 134.

Also part of the invention are polynucleotide sequences that encode thenovel anti-human NGF human antibodies, vectors comprising thepolynucleotide sequences encoding anti-human NGF human antibodies, hostcells transformed with vectors incorporating polynucleotides that encodethe anti-human NGF human antibodies, formulations comprising anti-humanNGF human antibodies and methods of making and using the same.

The invention also provides methods for detecting the level of NGF in abiological sample, comprising the step of contacting the sample with anantibody of the invention or antigen-binding fragment thereof. Ananti-NGF antibody of the invention may be employed in any known assaymethod, such as competitive binding assays, direct and indirect sandwichassays, immunoprecipitation assays and enzyme-linked immunosorbentassays (ELISA) (See, Sola, 1987, Monoclonal Antibodies: A Manual ofTechniques, pp. 147-158, CRC Press, Inc.) for the detection andquantitation of NGF. The antibodies can bind NGF with an affinity thatis appropriate for the assay method being employed.

In addition, the invention provides methods for treating a diseaseassociated with increased production of NGF, or increased sensitivity toNGF comprising the step of administering a pharmaceutically effectiveamount of a pharmaceutical composition comprising at least one antibodyof the invention or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof to an individual in need thereof.

In certain embodiments, the invention relates to a method of treating acondition caused by increased expression of nerve growth factor (NGF) orincreased sensitivity to NGF comprising administering to a patientorally, through injection by intravenous, intraperitoneal, intracerebral(intra-parenchymal), intracerebroventricular, intramuscular,intra-ocular, intraarterial, intraportal, intralesional or subcutaneousroutes, by sustained release systems or by implantation devices apharmaceutically effective amount of an NGF antibody., wherein thecondition is acute pain, dental pain, pain from trauma, surgical pain,pain resulting from amputation or abscess, causalgia, demyelinatingdiseases, trigeminal neuralgia, cancer, chronic alcoholism, stroke,thalamic pain syndrome, diabetes, acquired immune deficiency syndrome(“AIDS”), toxins, chemotherapy, general headache, migraine, clusterheadache, mixed-vascular or non-vascular syndromes, tension headache,general inflammation, arthritis, rheumatic diseases, lupus,osteoarthritis, fibromyalgia, inflammatory bowel disorders, irritablebowel syndrome, inflammatory eye disorders, inflammatory or unstablebladder disorders, psoriasis, skin complaints with inflammatorycomponents, sunburn, carditis, dermatitis, myositis, neuritis, collagenvascular diseases, chronic inflammatory conditions, inflammatory painand associated hyperalgesia and allodynia, neuropathic pain andassociated hyperalgesia or allodynia, diabetic neuropathy pain,causalgia, sympathetically maintained pain, deafferentation syndromes,asthma, epithelial tissue damage or dysfunction, herpes simplex,disturbances of visceral motility at respiratory, genitourinary,gastrointestinal or vascular regions, wounds, burns, allergic skinreactions, pruritis, vitiligo, general gastrointestinal disorders,colitis, gastric ulceration, duodenal ulcers, vasomotor or allergicrhinitis, or bronchial disorders, dysmenorrhoea, dyspepsia,gastroesophageal reflux, pancreatitis, or visceralgia.

In certain embodiments, the methods comprise a pharmaceuticallyeffective amount of an NGF antibody and are useful for treating orpreventing osteoarthritis knee pain. In certain embodiments thepharmaceutically effective amount an NGF antibody is from about 3 mg toabout 30 mg per subcutaneous injection. In certain embodiments theadministration comprises multiple subcutaneous injections. Inembodiments, the administration comprises a single subcutaneousinjection. In certain embodiments, the compositions and methods of theinvention comprise an NGF antibody comprising a light chain comprisingSEQ ID NO. 44. In certain embodiments, the NGF antibody comprises aheavy chain comprising SEQ ID. NO. 40. In further embodiments, the NGFantibody comprises a light chain comprising SEQ ID NO. 44, and a heavychain comprising SEQ ID. NO. 40.

Accordingly, in accordance with the above description of the invention,in various aspects the invention relates to methods and compositionscomprising an NGF antibody comprising a light chain comprising SEQ IDNO: 44 and a heavy chain comprising SEQ ID NO: 40, wherein the heavychain and light chain of the antibody are connected by a flexible linkerto form a single chain antibody. In some embodiments of this aspect, theNGF antibody comprises a single-chain Fv antibody, a Fab′ antibody, a(Fab′)₂ antibody, a fully human antibody, and/or a humanized antibody.In some embodiments of this aspect the NGF antibody inhibits NGFsignaling.

In certain embodiments of this aspect, the NGF antibody dissociates froma human NGF polypeptide with a K_(D) of about 1×10⁻⁹ or less, about1×10⁻¹⁰ or less, or about 1×10⁻¹¹ or less. In certain embodiments ofthis aspect, the NGF antibody neutralizes human NGF bioactivity in astandard in vitro assay with an IC₅₀ of about 1×10⁻⁸ or less, about1×10⁻⁹ or less, or about 0.2×10⁻⁹ or less. In certain embodiments, theNGF antibody dissociates from a human NGF polypeptide with theabove-mentioned K_(D) value(s) and neutralizes human NGF bioactivity ina standard in vitro assay with the above-mentioned IC₅₀ values.

Specific preferred embodiments of the invention will become evident fromthe following more detailed description of certain preferred embodimentsand the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1( a)-(b) depict graphs that demonstrate neutralization of NGFactivity in the DRG neuron based neutralization bioassay by 4D4monoclonal antibodies purified from the hybridoma conditioned media.

FIG. 2 depicts graphs that demonstrate VR1 expression stimulated byhuman NGF activity and neutralization of NGF activity in DRG neuronbased neutralization bioassays by an anti-NGF monoclonal antibody (4D4)purified from the hybridoma conditioned media.

FIG. 3 depicts graphs that demonstrate neutralization of NGF activity inDRG neuron based neutralization bioassays by transiently expressedrecombinant anti-NGF 4D4 monoclonal antibodies when expressed as eitheran IgG1 or IgG2 and in cells grown either in a roller bottle culture (R)or in spinner flasks (S).

FIG. 4 depicts sequence alignments of neurotrophins. The numbering andsecondary structure elements above the sequence refer to mature humanNGF. Conserved residues are marked with a star, and regions with lowsequence homology are shaded. NGF human is SEQ ID NO: 135; NGF mouse isSEQ ID NO: 136; BDNF is SEQ ID NO: 137; NT3 is SEQ ID NO: 138.

FIG. 5 shows anti-NGF CDR1 heavy chain alignment and percent identityfor the 14D10 (SEQ ID NO: 98), 6H9 (SEQ ID NO: 104), 7H2 (SEQ ID NO:110), 4G6 (SEQ ID NO: 116), 14D11 (SEQ ID NO: 92), and 4D4 (SEQ ID NO:22) antibodies.

FIG. 6 shows anti-NGF CDR2 heavy chain alignment and percent identityfor the 14D10 (SEQ ID NO: 99), 6H9 (SEQ ID NO: 105), 7H2 (SEQ ID NO:111), 4G6 (SEQ ID NO: 117), 14D11 (SEQ ID NO: 93), and 4D4 (SEQ ID NO:18) antibodies.

FIG. 7 shows anti-NGF CDR3 heavy chain alignment and percent identityfor the 14D10 (SEQ ID NO: 100), 6H9 (SEQ ID NO: 106), 7H2 (SEQ ID NO:112), 4G6 (SEQ ID NO: 118), 14D11 (SEQ ID NO: 94), and 4D4 (SEQ ID NO:14) antibodies.

FIG. 8 shows anti-NGF CDR1 light chain alignment and percent identityfor the 14D10 (SEQ ID NO: 95), 6H9 (SEQ ID NO: 107), 7H2 (SEQ ID NO:113), 4G6a (SEQ ID NO: 119), 4G6b (SEQ ID NO: 122), 4G6c (SEQ ID NO:125), 4G6d (SEQ ID NO: 128), 4G6e (SEQ ID NO: 132), 14D11 (SEQ ID NO:95), and 4D4 (SEQ ID NO: 24) antibodies (4G6a is referred to in variousFigures as 20031028340; 4G6b is referred to in various Figures as20031028351; 4G6c is referred to in various Figures as 20031071526; 4G6dis referred to in various Figures as 20031028344; 4G6e is referred to invarious Figures as 20031000528).

FIG. 9 shows anti-NGF CDR2 light chain alignment and percent identityfor the 14D10 (SEQ ID NO: 96), 6H9 (SEQ ID NO: 108), 7H2 (SEQ ID NO:114), 4G6a (SEQ ID NO: 120), 4G6b (SEQ ID NO: 123), 4G6c (SEQ ID NO:126), 4G6d (SEQ ID NO: 129), 4G6e (SEQ ID NO: 133), 14D11 (SEQ ID NO:96), and 4D4 (SEQ ID NO: 20) antibodies (4G6a is referred to in variousFigures as 20031028340; 4G6b is referred to in various Figures as20031028351; 4G6c is referred to in various Figures as 20031071526; 4G6dis referred to in various Figures as 20031028344; 4G6e is referred to invarious Figures as 20031000528).

FIG. 10 shows anti-NGF CDR3 light chain alignment and percent identityfor the 14D10 (SEQ ID NO: 97), 6H9 (SEQ ID NO: 109), 7H2 (SEQ ID NO:115), 4G6a (SEQ ID NO: 121), 4G6b (SEQ ID NO: 124), 4G6c (SEQ ID NO:127), 4G6d (SEQ ID NO: 130), 4G6e (SEQ ID NO: 134), 14D11 (SEQ ID NO:97), and 4D4 (SEQ ID NO: 16) antibodies (4G6a is referred to in variousFigures as 20031028340; 4G6b is referred to in various Figures as20031028351; 4G6c is referred to in various Figures as 20031071526; 4G6dis referred to in various Figures as 20031028344; 4G6e is referred to invarious Figures as 20031000528).

FIG. 11 shows anti-NGF light chain alignment and percent identity forthe 14D10 (SEQ ID NO: 82), 6H9 (SEQ ID NO: 84), 7H2 (SEQ ID NO: 86),4G6a (SEQ ID NO: 88), 4G6b (SEQ ID NO: 89), 4G6c (SEQ ID NO: 90), 4G6d(SEQ ID NO: 91), 4G6e (SEQ ID NO: 131), 14D11 (SEQ ID NO: 80), and 4D4(SEQ ID NO: 12) antibodies (4G6a is referred to in various Figures as20031028340; 4G6b is referred to in various Figures as 20031028351; 4G6cis referred to in various Figures as 20031071526; 4G6d is referred to invarious Figures as 20031028344; 4G6e is referred to in various Figuresas 20031000528).

FIG. 12 shows anti-NGF heavy chain alignment and percent identity forthe 4D4 (SEQ ID NO: 10), 4G6 (SEQ ID NO: 87), 14D10 (SEQ ID NO: 81),14D11 (SEQ ID NO: 79), 7H2 (SEQ ID NO: 85), and 6H9 (SEQ ID NO: 83)antibodies.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All references cited in this application are expressly incorporated byreference herein for any purpose.

Definitions

Conventional techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures may be generally performed according to methods wellknown in the art and as described in various general and more specificreferences that are cited and discussed throughout the presentspecification. See e.g., Sambrook et al., 2001, MOLECULAR CLONING: ALABORATORY MANUAL, 3d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., which is incorporated herein by reference for anypurpose. Unless specific definitions are provided, the nomenclatureutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are those wellknown and commonly used in the art. Similarly, conventional techniquesmay be used for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings: The phrases “biological property”, “biologicalcharacteristic”, and the term “activity” in reference to an antibody ofthe present invention are used interchangeably herein and include, butare not limited to, epitope affinity and specificity (e.g., anti-humanNGF human antibody binding to human NGF), ability to antagonize theactivity of the targeted polypeptide (e.g., NGF activity), the in vivostability of the antibody, and the immunogenic properties of theantibody. Other identifiable biological properties or characteristics ofan antibody recognized in the art include, for example,cross-reactivity, (i.e., with non-human homologs of the targetedpolypeptide, or with other proteins or tissues, generally), and abilityto preserve high expression levels of protein in mammalian cells. Theaforementioned properties or characteristics can be observed or measuredusing art-recognized techniques including, but not limited to ELISA,competitive ELISA, surface plasmon resonance analysis, in vitro and invivo neutralization assays (e.g., Example 2), and immunohistochemistrywith tissue sections from different sources including human, primate, orany other source as the need may be. Particular activities andbiological properties of anti-human NGF human antibodies are describedin further detail in the Examples below.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the isolated polynucleotide (1)is not associated with all or a portion of a polynucleotide in which theisolated polynucleotide is found in nature, (2) is linked to apolynucleotide to which it is not linked in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “isolated protein” referred to herein means that a subjectprotein (1) is free of at least some other proteins with which it wouldnormally be found, (2) is essentially free of other proteins from thesame source, e.g., from the same species, (3) is expressed by a cellfrom a different species, (4) has been separated from at least about 50percent of polynucleotides, lipids, carbohydrates, or other materialswith which it is associated in nature, (5) is not associated (bycovalent or noncovalent interaction) with portions of a protein withwhich the “isolated protein” is associated in nature, (6) is operablyassociated (by covalent or noncovalent interaction) with a polypeptidewith which it is not associated in nature, or (7) does not occur innature. Such an isolated protein can be encoded by genomic DNA, cDNA,mRNA or other RNA, of synthetic origin, or any combination thereof.Preferably, the isolated protein is substantially free from proteins orpolypeptides or other contaminants that are found in its naturalenvironment that would interfere with its use (therapeutic, diagnostic,prophylactic, research or otherwise).

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or theapeutic uses for the antibody, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the antibody willbe purified (1) to greater than 95% by weight of antibody as determinedby the Lowry method, and most preferably more than 99% by weight, (2) toa degree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.

The terms “polypeptide” or “protein” means molecules having the sequenceof native proteins, that is, proteins produced by naturally-occurringand specifically non-recombinant cells, or genetically-engineered orrecombinant cells, and comprise molecules having the amino acid sequenceof the native protein, or molecules having deletions from, additions to,and/or substitutions of one or more amino acids of the native sequence.The terms “polypeptide” and “protein” specifically encompass anti-NGFantibodies, or sequences that have deletions from, additions to, and/orsubstitutions of one or more amino acid of an anti-NGF antibody.

The term “polypeptide fragment” refers to a polypeptide that has anamino-terminal deletion, a carboxyl-terminal deletion, and/or aninternal deletion. In certain embodiments, fragments are at least 5 toabout 500 amino acids long. It will be appreciated that in certainembodiments, fragments are at least 5, 6, 8, 10, 14, 20, 50, 70, 100,110, 150, 200, 250, 300, 350, 400, or 450 amino acids long. Particularlyuseful polypeptide fragments include functional domains, includingbinding domains. In the case of an anti-NGF antibody, useful fragmentsinclude but are not limited to a CDR region, a variable domain of aheavy or light chain, a portion of an antibody chain or just itsvariable region including two CDRs, and the like.

The term “specific binding agent” refers to a natural or non-naturalmolecule that specifically binds to a target. Examples of specificbinding agents include, but are not limited to, proteins, peptides,nucleic acids, carbohydrates, and lipids. In certain embodiments, aspecific binding agent is an antibody.

The term “specific binding agent to NGF” refers to a specific bindingagent that specifically binds any portion of NGF. In certainembodiments, a specific binding agent to NGF is an antibody that bindsspecifically to NGF.

The term “immunologically functional immunoglobulin fragment” as usedherein refers to a polypeptide fragment that contains at least the CDRsof the immunoglobulin heavy and light chains. An immunologicallyfunctional immunoglobulin fragment of the invention is capable ofbinding to an antigen. In preferred embodiments, the antigen is a ligandthat specifically binds to a receptor. In these embodiments, binding ofan immunologically functional immunoglobulin fragment of the inventionprevents binding of the ligand to its receptor, interrupting thebiological response resulting from ligand binding to the receptor.Preferably, an immunologically functional immunoglobulin fragment of theinvention binds specifically to NGF. Most preferably, the fragment bindsspecifically to human NGF.

The term “naturally-occurring” as used herein and applied to an objectrefers to the fact that the object can be found in nature. For example,a polypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andthat has not been intentionally modified by man is naturally-occurring.

The term “operably linked” means that the components to which the termis applied are in a relationship that allows them to carry out theirinherent functions under suitable conditions. For example, a controlsequence “operably linked” to a protein coding sequence is ligatedthereto so that expression of the protein coding sequence is achievedunder conditions compatible with the transcriptional activity of thecontrol sequences.

The term “control sequence” as used herein refers to polynucleotidesequences that can effect expression, processing or intracellularlocalization of coding sequences to which they are ligated. The natureof such control sequences may depend upon the host organism. Inparticular embodiments, control sequences for prokaryotes may include apromoter, ribosomal binding site, and transcription terminationsequence. In other particular embodiments, control sequences foreukaryotes may include promoters comprising one or a plurality ofrecognition sites for transcription factors, transcription enhancersequences, transcription termination sequences and polyadenylationsequences. In certain embodiments, “control sequences” can includeleader sequences and/or fusion partner sequences.

The term “polynucleotide” as referred to herein means single-stranded ordouble-stranded nucleic acid polymers of at least 10 nucleotides inlength. In certain embodiments, the nucleotides comprising thepolynucleotide can be ribonucleotides or deoxyribonucleotides or amodified form of either type of nucleotide. Said modifications includebase modifications such as bromuridine, ribose modifications such asarabinoside and 2′,3′-dideoxyribose and intemucleotide linkagemodifications such as phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phoshoraniladate and phosphoroamidate. The term “polynucleotide”specifically includes single and double stranded forms of DNA.

The term “oligonucleotide” referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and/or non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset comprising members that aregenerally single-stranded and have a length of 200 nucleotides or fewer.In certain embodiments, oligonucleotides are 10 to 60 nucleotides inlength. In certain embodiments, oligonucleotides are 12, 13, 14, 15, 16,17, 18, 19, or 20 to 40 nucleotides in length. Oligonucleotides may besingle stranded or double stranded, e.g. for use in the construction ofa genetic mutant. Oligonucleotides of the invention may be sense orantisense oligonucleotides with reference to a protein-coding sequence.

The term “naturally occurring nucleotides” includes deoxyribonucleotidesand ribonucleotides. The term “modified nucleotides” includesnucleotides with modified or substituted sugar groups and the like. Theterm “oligonucleotide linkages” includes oligonucleotide linkages suchas phosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate, and the like. See, e.g., LaPlanche et al., 1986, Nucl.Acids Res., 14:9081; Stec et al., 1984, J. Am. Chem. Soc., 106:6077;Stein et al., 1988, Nucl. Acids Res., 16:3209; Zon et al., 1991,Anti-Cancer Drug Design, 6:539; Zon et al., 1991, OLIGONUCLEOTIDES ANDANALOGUES: A PRACTICAL APPROACH, pp. 87-108 (F. Eckstein, Ed.), OxfordUniversity Press, Oxford England; Stec et al., U.S. Pat. No. 5,151,510;Uhlmann and Peyman, 1990, Chemical Reviews, 90:543, the disclosures ofwhich are hereby incorporated by reference for any purpose. Anoligonucleotide can include a detectable label to enable detection ofthe oligonucleotide or hybridization thereof.

The term “vector” includes a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “expressionvectors”). In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The phrase “recombinant host cell” (or simply “host cell”) includes acell into which a recombinant expression vector has been introduced. Itwill be understood by those of skill in the art that such terms areintended to refer not only to the particular subject cell but to theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell but are still included within the scope of the term “host cell” asused herein. A wide variety of host expression systems can be used toexpress the antibodies of the present invention including bacterial,yeast, baculoviral and mammalian expression systems (as well as phagedisplay expression systems). An example of a suitable bacterialexpression vector is pUC19. To express an antibody recombinantly, a hostcell is transfected with one or more recombinant expression vectorscarrying DNA fragments encoding the immunoglobulin light and heavychains of the antibody such that the light and heavy chains areexpressed in the host cell and, preferably, secreted into the medium inwhich the host cells are cultured, from which medium the antibodies canbe recovered. Standard recombinant DNA methodologies are used to obtainantibody heavy and light chain genes, incorporate these genes intorecombinant expression vectors and introduce the vectors into hostcells, such as those described in Sambrook et al., 2001, MOLECULARCLONING, A LABORATORY MANUAL, Cold Spring Harbor Laboratories, Ausubel,F. M. et al. (eds.) Current Protocols in Molecular Biology, GreenePublishing Associates, (1989) and in U.S. Pat. No. 4,816,397 to Boss etal.

The term “host cell” is used to refer to a cell which has beentransformed, or is capable of being transformed with a nucleic acidsequence and then of expressing a selected gene of interest. The termincludes the progeny of the parent cell, whether or not the progeny isidentical in morphology or in genetic make-up to the original parent, solong as the selected gene is present.

The term “transduction” is used to refer to the transfer of genes fromone bacterium to another, usually by a phage. “Transduction” also refersto the acquisition and transfer of eukaryotic cellular sequences byretroviruses.

The term “transfection” is used to refer to the uptake of foreign orexogenous DNA by a cell, and a cell has been “transfected” when theexogenous DNA has been introduced inside the cell membrane. A number oftransfection techniques are well known in the art and are disclosedherein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook etal., 2001, MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring HarborLaboratories; Davis et al., 1986, BASIC METHODS IN MOLECULAR BIOLOGY,Elsevier; and Chu et al., 1981, Gene 13:197. Such techniques can be usedto introduce one or more exogenous DNA moieties into suitable hostcells.

The term “transformation” as used herein refers to a change in a cell'sgenetic characteristics, and a cell has been transformed when it hasbeen modified to contain a new DNA. For example, a cell is transformedwhere it is genetically modified from its native state. Followingtransfection or transduction, the transforming DNA may recombine withthat of the cell by physically integrating into a chromosome of thecell, or may be maintained transiently as an episomal element withoutbeing replicated, or may replicate independently as a plasmid. A cell isconsidered to have been stably transformed when the DNA is replicatedwith the division of the cell.

The term “naturally occurring” or “native” when used in connection withbiological materials such as nucleic acid molecules, polypeptides, hostcells, and the like, refers to materials which are found in nature andare not manipulated by man. Similarly, “non-naturally occurring” or“non-native” as used herein refers to a material that is not found innature or that has been structurally modified or synthesized by man.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as anantibody, and additionally capable of being used in an animal to produceantibodies capable of binding to an epitope of that antigen. An antigenmay have one or more epitopes.

The term “identity,” as known in the art, refers to a relationshipbetween the sequences of two or more polypeptide molecules or two ormore nucleic acid molecules, as determined by comparing the sequencesthereof. In the art, “identity” also means the degree of sequencerelatedness between nucleic acid molecules or polypeptides, as the casemay be, as determined by the match between strings of two or morenucleotide or two or more amino acid sequences. “Identity” measures thepercent of identical matches between the smaller of two or moresequences with gap alignments (if any) addressed by a particularmathematical model or computer program (i.e., “algorithms”).

The term “similarity” is used in the art with regard to a relatedconcept, but in contrast to “identity,” “similarity” refers to a measureof relatedness, which includes both identical matches and conservativesubstitution matches. If two polypeptide sequences have, for example,10/20 identical amino acids, and the remainder are all non-conservativesubstitutions, then the percent identity and similarity would both be50%. If in the same example, there are five more positions where thereare conservative substitutions, then the percent identity remains 50%,but the percent similarity would be 75% (15/20). Therefore, in caseswhere there are conservative substitutions, the percent similaritybetween two polypeptides will be higher than the percent identitybetween those two polypeptides.

Identity and similarity of related nucleic acids and polypeptides can bereadily calculated by known methods. Such methods include, but are notlimited to, those described in COMPUTATIONAL MOLECULAR BIOLOGY, (Lesk,A. M., ed.), 1988, Oxford University Press, New York; BIOCOMPUTING:INFORMATICS AND GENOME PROJECTS, (Smith, D. W., ed.), 1993, AcademicPress, New York; COMPUTER ANALYSIS OF SEQUENCE DATA, Part 1, (Griffin,A. M., and Griffin, H. G., eds.), 1994, Humana Press, New Jersey; vonHeinje, G., SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, 1987, AcademicPress; SEQUENCE ANALYSIS PRIMER, (Gribskov, M. and Devereux, J., eds.),1991, M. Stockton Press, New York; Carillo et al., 1988, SIAM J. AppliedMath., 48:1073; and Durbin et al., 1998, BIOLOGICAL SEQUENCE ANALYSIS,Cambridge University Press.

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity aredescribed in publicly available computer programs. Preferred computerprogram methods to determine identity between two sequences include, butare not limited to, the GCG program package, including GAP (Devereux etal., 1984, Nucl. Acid. Res., 12:387; Genetics Computer Group, Universityof Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul etal., 1990, J. Mol Biol., 215:403-410). The BLASTX program is publiclyavailable from the National Center for Biotechnology Information (NCBI)and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda,Md. 20894; Altschul et al., 1990, supra). The well-known Smith Watermanalgorithm may also be used to determine identity.

Certain alignment schemes for aligning two amino acid sequences mayresult in matching of only a short region of the two sequences, and thissmall aligned region may have very high sequence identity even thoughthere is no significant relationship between the two full-lengthsequences. Accordingly, in certain embodiments, the selected alignmentmethod (GAP program) will result in an alignment that spans at least 50contiguous amino acids of the target polypeptide.

For example, using the computer algorithm GAP (Genetics Computer Group,University of Wisconsin, Madison, Wis.), two polypeptides for which thepercent sequence identity is to be determined are aligned for optimalmatching of their respective amino acids (the “matched span”, asdetermined by the algorithm). In certain embodiments, a gap openingpenalty (which is calculated as three-times the average diagonal; wherethe “average diagonal” is the average of the diagonal of the comparisonmatrix being used; the “diagonal” is the score or number assigned toeach perfect amino acid match by the particular comparison matrix) and agap extension penalty (which is usually one-tenth of the gap openingpenalty), as well as a comparison matrix such as PAM250 or BLOSUM 62 areused in conjunction with the algorithm. In certain embodiments, astandard comparison matrix (see Dayhoff et al., 1978, Atlas of ProteinSequence and Structure, 5:345-352 for the PAM 250 comparison matrix;Henikoff et al., 1992, Proc. Natl. Acad. Sci USA, 89:10915-10919 for theBLOSUM 62 comparison matrix) is also used by the algorithm.

In certain embodiments, the parameters for a polypeptide sequencecomparison include the following:

-   -   Algorithm: Needleman et al., 1970, J. Mol. Biol., 48:443-453;    -   Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra;    -   Gap Penalty: 12    -   Gap Length Penalty: 4    -   Threshold of Similarity: 0        The GAP program may be useful with the above parameters. In        certain embodiments, the aforementioned parameters are the        default parameters for polypeptide comparisons (along with no        penalty for end gaps) using the GAP algorithm.

The term “homology” refers to the degree of similarity between proteinor nucleic acid sequences. Homology information is useful for theunderstanding the genetic relatedness of certain protein or nucleic acidspecies. Homology can be determined by aligning and comparing sequences.Typically, to determine amino acid homology, a protein sequence iscompared to a database of known protein sequences. Homologous sequencesshare common functional identities somewhere along their sequences. Ahigh degree of similarity or identity is usually indicative of homology,although a low degree of similarity or identity does not necessarilyindicate lack of homology.

Several approaches can be used to compare amino acids from one sequenceto amino acids of another sequence to determine homology. Generally, theapproaches fall into two categories: (1) comparison of physicalcharacteristics such as polarity, charge, and Van der Waals volume, togenerate a similarity matrix; and (2) comparison of likely substitutionof an amino acid in a sequence by any other amino acid, which is basedon observation of many protein sequences from known homologous proteinsand to generate a Point Accepted Mutation Matrix (PAM).

The percentage of identity may also be calculated by using the programneedle (EMBOSS package) or stretcher (EMBOSS package) or the programalign X, as a module of the vector NTI suite 9.0.0 software package,using the default parameters (for example, GAP penalty 5, GAP openingpenalty 15, GAP extension penalty 6.6).

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See IMMUNOLOGY—A SYNTHESIS, 2ndEdition, (E. S. Golub and D. R. Gren, Eds.), Sinauer Associates:Sunderland, Mass., 1991, incorporated herein by reference for anypurpose. Stereoisomers (e.g., D-amino acids) of the twenty conventionalamino acids; unnatural amino acids such as α-, α-disubstituted aminoacids, N-alkyl amino acids, lactic acid, and other unconventional aminoacids may also be suitable components for polypeptides of the invention.Examples of unconventional amino acids include: 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, σ-N-methylarginine, and other similar amino acids andimino acids (e.g., 4-hydroxyproline). In the polypeptide notation usedherein, the left-hand direction is the amino terminal direction and theright-hand direction is the carboxyl-terminal direction, in accordancewith standard usage and convention.

Naturally occurring residues may be divided into classes based on commonside chain properties:

-   -   1) hydrophobic: norleucine (Nor), Met, Ala, Val, Leu, Ile, Phe,        Trp, Tyr, Pro;    -   2) polar hydrophilic: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser,        Thr;    -   3) aliphatic: Ala, Gly, Ile, Leu, Val, Pro;    -   4) aliphatic hydrophobic: Ala, Ile, Leu, Val, Pro;    -   5) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   6) acidic: Asp, Glu;    -   7) basic: His, Lys, Arg;    -   8) residues that influence chain orientation: Gly, Pro;    -   9) aromatic: His, Trp, Tyr, Phe; and    -   10) aromatic hydrophobic : Phe, Trp, Tyr.

Conservative amino acid substitutions may involve exchange of a memberof one of these classes with another member of the same class.Conservative amino acid substitutions may encompass non-naturallyoccurring amino acid residues, which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics and other reversed or invertedforms of amino acid moieties.

Non-conservative substitutions may involve the exchange of a member ofone of these classes for a member from another class. Such substitutedresidues may be introduced into regions of the human antibody that arehomologous with non-human antibodies, or into the non-homologous regionsof the molecule.

In making such changes, according to certain embodiments, thehydropathic index of amino acids may be considered. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics. They are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art(see, for example, Kyte et al., 1982, J. Mol. Biol. 157:105-131). It isknown that certain amino acids may be substituted for other amino acidshaving a similar hydropathic index or score and still retain a similarbiological activity. In making changes based upon the hydropathic index,in certain embodiments, the substitution of amino acids whosehydropathic indices are within ±2 is included. In certain embodiments,those that are within ±1 are included, and in certain embodiments, thosewithin ±0.5 are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments, asdisclosed herein. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within ±2 is included, in certainembodiments, those that are within ±1 are included, and in certainembodiments, those within ±0.5 are included. One may also identifyepitopes from primary amino acid sequences on the basis ofhydrophilicity. These regions are also referred to as “epitopic coreregions.”

Exemplary amino acid substitutions are set forth in Table 1.

TABLE 1 Amino Acid Substitutions Original Preferred Residues ExemplarySubstitutions Substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn LysAsn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp GlyPro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Leu Phe,Norleucine Leu Norleucine, Ile, Val, Met, Ile Ala, Phe Lys Arg, 1,4Diamino-butyric Arg Acid, Gln, Asn Met Leu, Phe, Ile Leu Phe Leu, Val,Ile, Ala, Tyr Leu Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr,Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Ala, LeuNorleucine

A skilled artisan will be able to determine suitable variants of thepolypeptide as set forth herein using well-known techniques. In certainembodiments, one skilled in the art may identify suitable areas of themolecule that may be changed without destroying activity by targetingregions not believed to be important for activity. In other embodiments,the skilled artisan can identify residues and portions of the moleculesthat are conserved among similar polypeptides. In further embodiments,even areas that may be important for biological activity or forstructure may be subject to conservative amino acid substitutionswithout destroying the biological activity or without adverselyaffecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, the skilledartisan can predict the importance of amino acid residues in a proteinthat correspond to amino acid residues important for activity orstructure in similar proteins. One skilled in the art may opt forchemically similar amino acid substitutions for such predicted importantamino acid residues.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of such information, one skilled in the art maypredict the alignment of amino acid residues of an antibody with respectto its three dimensional structure. In certain embodiments, one skilledin the art may choose to not make radical changes to amino acid residuespredicted to be on the surface of the protein, since such residues maybe involved in important interactions with other molecules. Moreover,one skilled in the art may generate test variants containing a singleamino acid substitution at each desired amino acid residue. The variantscan then be screened using activity assays known to those skilled in theart. Such variants could be used to gather information about suitablevariants. For example, if one discovered that a change to a particularamino acid residue resulted in destroyed, undesirably reduced, orunsuitable activity, variants with such a change can be avoided. Inother words, based on information gathered from such routineexperiments, one skilled in the art can readily determine the aminoacids where further substitutions should be avoided either alone or incombination with other mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See Moult, 1996, Curr. Op. in Biotech.7:422-427; Chou et al., 1974, Biochemistry 13:222-245; Chou et al.,1974, Biochemistry 113:211-222; Chou et al., 1978, Adv. Enzymol. Relat.Areas Mol. Biol. 47:45-148; Chou et al., 1979, Ann. Rev. Biochem.47:251-276; and Chou et al., 1979, Biophys. J. 26:367-384. Moreover,computer programs are currently available to assist with predictingsecondary structure. One method of predicting secondary structure isbased upon homology modeling. For example, two polypeptides or proteinsthat have a sequence identity of greater than 30%, or similarity greaterthan 40% often have similar structural topologies. The recent growth ofthe protein structural database (PDB) has provided enhancedpredictability of secondary structure, including the potential number offolds within a polypeptide's or protein's structure. See Holm et al.,1999, Nucl. Acid. Res. 27:244-247. It has been suggested (Brenner etal., 1997, Curr. Op. Struct. Biol. 7:369-376) that there are a limitednumber of folds in a given polypeptide or protein and that once acritical number of structures have been resolved, structural predictionwill become dramatically more accurate.

Additional methods of predicting secondary structure include “threading”(Jones, 1997, Curr. Opin. Struct. Biol. 7:377-87; Sippl et al., 1996,Structure 4:15-19), “profile analysis” (Bowie et al., 1991, Science253:164-170; Gribskov et al., 1990, Meth. Enzym. 183:146-159; Gribskovet al., 1987, Proc. Nat. Acad. Sci. 84:4355-4358), and “evolutionarylinkage” (See Holm, 1999, supra; and Brenner, 1997, supra).

In certain embodiments, antibody variants include glycosylation variantswherein the number and/or type of glycosylation site has been alteredcompared to the amino acid sequences of the parent polypeptide. Incertain embodiments, protein variants comprise a greater or a lessernumber of N-linked glycosylation sites than the native protein. AnN-linked glycosylation site is characterized by the sequence: Asn-X-Seror Asn-X-Thr, wherein the amino acid residue designated as X may be anyamino acid residue except proline. The substitution of amino acidresidues to create this sequence provides a potential new site for theaddition of an N-linked carbohydrate chain. Alternatively, substitutionsthat eliminate this sequence will remove an existing N-linkedcarbohydrate chain. Also provided is a rearrangement of N-linkedcarbohydrate chains wherein one or more N-linked glycosylation sites(typically those that are naturally occurring) are eliminated and one ormore new N-linked sites are created. Additional preferred antibodyvariants include cysteine variants wherein one or more cysteine residuesare deleted from or substituted for another amino acid (e.g., serine)compared to the parent amino acid sequence. Cysteine variants may beuseful when antibodies must be refolded into a biologically activeconformation such as after the isolation of insoluble inclusion bodies.Cysteine variants generally have fewer cysteine residues than the nativeprotein, and typically have an even number to minimize interactionsresulting from unpaired cysteines.

In additional embodiments, antibody variants can include antibodiescomprising a modified Fc fragment or a modified heavy chain constantregion. An Fc fragment, which stands for “fragment that crystallizes,”or a heavy chain constant region can be modified by mutation to conferon an antibody altered binding characteristics. See, for example, Burtonand Woof, 1992, Advances in Immunology 51: 1-84; Ravetch and Bolland,2001, Annu. Rev. Immunol. 19: 275-90; Shields et al., 2001, Journal ofBiol. Chem 276: 6591-6604; Telleman and Junghans, 2000, Immunology 100:245-251; Medesan et al., 1998, Eur. J. Immunol 28: 2092-2100; all ofwhich are incorporated herein by reference). Such mutations can includesubstitutions, additions, deletions, or any combination thereof, and aretypically produced by site-directed mutagenesis using one or moremutagenic oligonucleotide(s) according to methods described herein, aswell as according to methods known in the art (see, for example,Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 3rd Ed., 2001,Cold Spring Harbor, N.Y. and Berger and Kimmel, METHODS IN ENZYMOLOGY,Volume 152, Guide to Molecular Cloning Techniques, 1987, Academic Press,Inc., San Diego, Calif., which are incorporated herein by reference).

According to certain embodiments, amino acid substitutions are thosethat: (1) reduce susceptibility to proteolysis, (2) reducesusceptibility to oxidation, (3) alter binding affinity for formingprotein complexes, (4) alter binding affinities, and/or (5) confer ormodify other physicochemical or functional properties on suchpolypeptides. According to certain embodiments, single or multiple aminoacid substitutions (in certain embodiments, conservative amino acidsubstitutions) may be made in the naturally occurring sequence (incertain embodiments, in the portion of the polypeptide outside thedomain(s) forming intermolecular contacts). In preferred embodiments, aconservative amino acid substitution typically does not substantiallychange the structural characteristics of the parent sequence (e.g., areplacement amino acid should not tend to break a helix that occurs inthe parent sequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in PROTEINS,STRUCTURES AND MOLECULAR PRINCIPLES, (Creighton, Ed.), 1984, W. H.Freeman and Company, New York; INTRODUCTION TO PROTEIN STRUCTURE (C.Branden and J. Tooze, eds.), 1991, Garland Publishing, New York, N.Y.;and Thornton et al., 1991, Nature 354:105, each of which areincorporated herein by reference.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics”. See Fauchere, 1986, Adv. Drug Res.15:29; Veber & Freidinger, 1985, TINS p. 392; and Evans et al,. 1987, J.Med. Chem. 30:1229, which are incorporated herein by reference for anypurpose. Such compounds are often developed with the aid of computerizedmolecular modeling. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce a similartherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i.e., a polypeptide thathas a biochemical property or pharmacological activity), such as humanantibody, but have one or more peptide linkages optionally replaced by alinkage selected from: —CH₂—NH—, —CH₂—S—, —CH₂—CH₂—, —CH═CH-(cis andtrans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, by methods well known in theart. Systematic substitution of one or more amino acids of a consensussequence with a D-amino acid of the same type (e.g., D-lysine in placeof L-lysine) may be used in certain embodiments to generate more stablepeptides. In addition, constrained peptides comprising a consensussequence or a substantially identical consensus sequence variation maybe generated by methods known in the art (Rizo & Gierasch, 1992, Ann.Rev. Biochem. 61:387, incorporated herein by reference for any purpose);for example, by adding internal cysteine residues capable of formingintramolecular disulfide bridges which cyclize the peptide.

“Antibody” or “antibody peptide(s)” refer to an intact antibody, or abinding fragment thereof that competes with the intact antibody forspecific binding. In certain embodiments, binding fragments are producedby recombinant DNA techniques. In additional embodiments, bindingfragments are produced by enzymatic or chemical cleavage of intactantibodies. Binding fragments include, but are not limited to, F(ab),F(ab′), F(ab′)₂, Fv, and single-chain antibodies.

The term “heavy chain” includes any immunoglobulin polypeptide havingsufficient variable region sequence to confer specificity for NGF. Theterm “light chain” includes any immunoglobulin polypeptide havingsufficient variable region sequence to confer specificity for NGF. Afull-length heavy chain includes a variable region domain, V_(H), andthree constant region domains, C_(H)1, C_(H)2, and C_(H)3. The V_(H)domain is at the amino-terminus of the polypeptide, and the C_(H)3domain is at the carboxyl-terminus. The term “heavy chain”, as usedherein, encompasses a full-length heavy chain and fragments thereof. Afull-length light chain includes a variable region domain, V_(L), and aconstant region domain, CL. Like the heavy chain, the variable regiondomain of the light chain is at the amino-terminus of the polypeptide.The term “light chain”, as used herein, encompasses a full-length lightchain and fragments thereof. A F(ab) fragment is comprised of one lightchain and the C_(H)1 and variable regions of one heavy chain. The heavychain of a F(ab) molecule cannot form a disulfide bond with anotherheavy chain molecule. A F(ab′) fragment contains one light chain and oneheavy chain that contains more of the constant region, between theC_(H)1 and C_(H)2 domains, such that an interchain disulfide bond can beformed between two heavy chains to form a F(ab′)₂ molecule. The Fvregion comprises the variable regions from both the heavy and lightchains, but lacks the constant regions. Single-chain antibodies are Fvmolecules in which the heavy and light chain variable regions have beenconnected by a flexible linker to form a single polypeptide chain, whichforms an antigen-binding region. Single chain antibodies are discussedin detail in International Patent Application Publication No. WO88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203.

A bivalent antibody other than a “multispecific” or “multifunctional”antibody, in certain embodiments, is understood to comprise bindingsites having identical antigenic specificity.

In assessing antibody binding and specificity according to theinvention, an antibody substantially inhibits adhesion of a ligand to areceptor when an excess of antibody reduces the quantity of ligand boundto receptor by at least about 20%, 40%, 60%, 80%, 85%, or more (asmeasured, inter alia, using an in vitro competitive binding assay).

By “neutralizing antibody” is meant an antibody molecule that is able toblock or substantially reduce an effector function of a target antigento which it binds. Accordingly, a “neutralizing” anti-NGF antibody iscapable of blocking or substantially reducing an effector function, suchas receptor binding and/or elicitation of a cellular response, of NGF.“Substantially reduce” is intended to mean at least about 60%,preferably at least about 70%, more preferably at least about 75%, evenmore preferably at least about 80%, still more preferably at least about85%, most preferably at least about 90% reduction of an effectorfunction of the target antigen (e.g., human NGF).

The term “epitope” includes any determinant, preferably a polypeptidedeterminant, capable of specific binding to an immunoglobulin or T-cellreceptor. In certain embodiments, epitope determinants includechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl groups, or sulfonyl groups, and, incertain embodiments, may have specific three-dimensional structuralcharacteristics, and/or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody. In certainembodiments, an antibody is said to specifically bind an antigen when itpreferentially recognizes its target antigen in a complex mixture ofproteins and/or macromolecules. In preferred embodiments, an antibody issaid to specifically bind an antigen when the equilibrium dissociationconstant is ≦10⁻⁸ M, more preferably when the equilibrium dissociationconstant is ≦10⁻⁹ M, and most preferably when the dissociation constantis ≦10⁻¹⁰ M.

An antibody binds “essentially the same epitope” as a referenceantibody, when the two antibodies recognize identical or stericallyoverlapping epitopes. The most widely used and rapid methods fordetermining whether two antibodies bind to identical or stericallyoverlapping epitopes are competition assays, which can be configured inall number of different formats, using either labeled antigen or labeledantibody. Usually, the antigen is immobilized on a substrate, and theability of unlabeled antibodies to block the binding of labeledantibodies is measured using radioactive or enzyme labels.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotin moieties that can bedetected by labeled avidin (e.g., streptavidin preferably comprising adetectable marker such as a fluorescent marker, a chemiluminescentmarker or an enzymatic activity that can be detected by optical orcolorimetric methods). In certain embodiments, the label can also betherapeutic. Various methods of labeling polypeptides and glycoproteinsare known in the art and may be used advantageously in the methodsdisclosed herein. Examples of labels for polypeptides include, but arenot limited to, the following: radioisotopes or radionuclides (eg ³H,¹⁴C, ¹⁵N ³⁵S, ⁹⁰Y, ^(99m)Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels(e.g., fluorescein isothiocyanate or FITC, rhodamine, or lanthanidephosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescentlabels, hapten labels such as biotinyl groups, and predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, or epitope tags). In certain embodiments, labels areattached by spacer arms (such as (CH₂)_(n), where n<about 20) of variouslengths to reduce potential steric hindrance.

The term “biological sample”, as used herein, includes, but is notlimited to, any quantity of a substance from a living thing or formerlyliving thing. Such living things include, but are not limited to,humans, mice, monkeys, rats, rabbits, and other animals. Such substancesinclude, but are not limited to, blood, serum, urine, cells, organs,tissues, bone, bone marrow, lymph nodes, and skin.

The term “pharmaceutical agent or drug” as used herein refers to achemical compound or composition capable of inducing a desiredtherapeutic effect when properly administered to a patient. Theexpression “pharmaceutically effective amount” in reference to apharmaceutical composition comprising one or a plurality of theantibodies of the invention is understood to mean, according to theinvention, an amount of the said pharmaceutical composition which iscapable of abolishing, in the patient considered, the decrease in thesensitivity threshold to external stimuli with a return of thissensitivity threshold to a level comparable to that observed in healthysubjects.

A “disorder” is any condition that would benefit from treatmentaccording to the present invention. “Disorder” and “condition” are usedinterchangeably herein and include chronic and acute NGF-mediateddisorders or NGF-mediated diseases, including those pathologicalconditions which predispose the mammal to the disorder in question.

The terms “NGF-mediated disease” and “NGF-mediated condition” encompassany medical condition or disorder associated with increased levels ofNGF or increased sensitivity to NGF including, but not limited to, acutepain, dental pain, pain from trauma, surgical pain, pain resulting fromamputation or abscess, causalgia, demyelinating diseases, trigeminalneuralgia, cancer, chronic alcoholism, stroke, thalamic pain syndrome,diabetes, acquired immune deficiency syndrome (“AIDS”), toxins andchemotherapy, general headache, migraine, cluster headache,mixed-vascular and non-vascular syndromes, tension headache, generalinflammation, arthritis, rheumatic diseases, lupus, osteoarthritis,inflammatory bowel disorders, irritable bowel syndrome, inflammatory eyedisorders, inflammatory or unstable bladder disorders, psoriasis, skincomplaints with inflammatory components, sunburn, carditis, dermatitis,myositis, neuritis, collagen vascular diseases, chronic inflammatoryconditions, inflammatory pain and associated hyperalgesia and allodynia,neuropathic pain and associated hyperalgesia and allodynia, diabeticneuropathy pain, causalgia, sympathetically maintained pain,deafferentation syndromes, asthma, epithelial tissue damage ordysfunction, herpes simplex, disturbances of visceral motility atrespiratory, genitourinary, gastrointestinal or vascular regions,wounds, burns, allergic skin reactions, pruritis, vitiligo, generalgastrointestinal disorders, colitis, gastric ulceration, duodenalulcers, vasomotor or allergic rhinitis, or bronchial disorders,dysmenorrhoea, dyspepsia, gastroesophageal reflux, pancreatitis, andvisceralgia.

As used herein, the terms “effective amount” and “therapeuticallyeffective amount” when used with reference to a vehicle- or apharmaceutical composition comprising one or more anti-human NGF humanantibody refers to an amount or dosage sufficient to produce a desiredresult (i.e., where for therapy with the vehicle- or anti-human NGFhuman antibodies of the present invention the desired result is thedesired reduction in inflammation and/or pain, for example) or tosupport an observable decrease in the level of one or more biologicalactivities of NGF. More specifically, a therapeutically effective amountis an amount of the anti-human NGF human antibody(ies) sufficient toinhibit, for some period of time, one or more of the clinically definedpathological processes associated with the condition at issue, e.g.,inflammation or pain, in a subject treated in vivo with the agent. Inthe present invention, an “effective amount” of an anti-NGF antibody mayprevent, stop, control, or reduce the perception of pain associated withany painful medical condition. In the methods of the present invention,the term “control” and grammatical variants thereof, are used to referto the prevention, partial or complete inhibition, reduction, delay orslowing down of an unwanted event, e.g., pain. The effective amount mayvary depending on the specific vehicle- or anti-human NGF humanantibody(ies) selected, and is also dependent on a variety of factorsand conditions related to the subject to be treated and the severity ofthe disorder. For example, if the vehicle- or anti-human NGF humanantibody(ies) is to be administered in vivo, factors such as the age,weight and health of the patient as well as dose response curves andtoxicity data obtained in preclinical animal work would be among thoseconsidered. If the agent is to be contacted with the cells in vitro, onewould also design a variety of pre-clinical in vitro studies to assesssuch parameters as uptake, half-life, dose, toxicity, etc. Thedetermination of an effective amount or a therapeutically effectiveamount for a given agent is well within the ability of those skilled inthe art.

As used herein, the terms “nerve growth factor” and “NGF” are defined asall mammalian species of native sequence NGF, including recombinanthuman NGF 1-120, shown as in SEQ ID NO:30.

As used herein, “substantially pure” or “substantially purified” means acompound or species that is the predominant species present (i.e., on amolar basis it is more abundant than any other individual species in thecomposition). In certain embodiments, a substantially purified fractionis a composition wherein the species comprises at least about 50 percent(on a molar basis) of all macromolecular species present. In certainembodiments, a substantially pure composition will comprise more thanabout 80%, 85%, 90%, 95%, or 99% of all macromolar species present inthe composition. In certain embodiments, the species is purified toessential homogeneity (contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single macromolecular species.

The term “patient” includes human and animal subjects. “Treatment” or“treat” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those prone to have the disorder or thosein which the disorder is to be prevented.

Unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

According to certain embodiments of the invention, antibodies directedto NGF may be used to treat neuropathic and inflammatory pain andNGF-mediated diseases, including but not limited to, those mentionedabove.

In one aspect of the invention are provided fully human monoclonalantibodies raised against and having biological and immunologicalspecificity for binding to human NGF. In another aspect the inventionprovides nucleic acids comprising nucleotide sequences encoding aminoacid sequences for heavy and light chain immunoglobulin molecules,particularly sequences corresponding to the variable regions thereof.Particular embodiments of this aspect of the invention are sequencescorresponding to complementarity determining regions (CDRs),specifically from CDR1 through CDR3, of the heavy and light chainsprovided by the invention. In yet another aspect the invention provideshybridoma cells and cell lines that express the immunoglobulin moleculesand antibodies, preferably monoclonal antibodies of the invention. Theinvention also provides biologically and immunologically purifiedpreparations of antibodies, preferably monoclonal antibodies raisedagainst and having biological and immunological specificity for bindingto human NGF.

The ability to clone and reconstruct megabase-sized human loci in yeastartificial chromosomes (YACs) and to introduce them into the mousegermline provides an advantageous approach to elucidating the functionalcomponents of very large or crudely mapped loci as well as generatinguseful models of human disease. Furthermore, the utilization of suchtechnology for substitution of mouse loci with their human equivalentsprovides unique insights into the expression and regulation of humangene products during development, their communication with othersystems, and their involvement in disease induction and progression.

An important practical application of such a strategy is the“humanization” of the mouse humoral immune system. Introduction of humanimmunoglobulin (Ig) loci into mice in which the endogenous Ig genes havebeen inactivated offers the opportunity to study mechanisms underlyingprogrammed expression and assembly of antibodies as well as their rolein B-cell development. Furthermore, such a strategy provides a sourcefor production of fully human monoclonal antibodies (MAbs).

The term “human antibody” includes antibodies having variable andconstant regions substantially corresponding to human germlineimmunoglobulin sequences. In certain embodiments, human antibodies areproduced in non-human mammals, including, but not limited to, rodents,such as mice and rats, and lagomorphs, such as rabbits. In certainembodiments, human antibodies are produced in hybridoma cells. Incertain embodiments, human antibodies are produced recombinantly.

The term “recombinant” in reference to an antibody includes antibodiesthat are prepared, expressed, created or isolated by recombinant means.Representative examples include antibodies expressed using a recombinantexpression vector transfected into a host cell, antibodies isolated froma recombinant, combinatorial human antibody library, antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (see e.g., Taylor, L. D., et al., Nucl. Acids Res.20:6287-6295,(1992); or antibodies prepared, expressed, created orisolated by any means that involves splicing of human immunoglobulingene sequences to other DNA sequences. Such recombinant human antibodieshave variable and constant regions derived from human germlineimmunoglobulin sequences.

Human antibodies have at least three advantages over non-human andchimeric antibodies for use in human therapy:

1) because the effector portion of the antibody is human, it mayinteract better with the other parts of the human immune system (e.g.,destroy the target cells more efficiently by complement-dependentcytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC));

2) the human immune system should not recognize the human antibody asforeign, and, therefore the antibody response against such an injectedantibody should be less than against a totally foreign non-humanantibody or a partially foreign chimeric antibody;

3) injected non-human antibodies have been reported to have a half-lifein the human circulation much shorter than the half-life of humanantibodies. Injected human antibodies will have a half-life essentiallyidentical to naturally occurring human antibodies, allowing smaller andless frequent doses to be given.

Thus, fully human antibodies are expected to minimize the immunogenicand allergic responses intrinsic to mouse or mouse-derivatized MAbs, andto thereby increase the efficacy and safety of the administeredantibodies. Fully human antibodies of the invention, therefore, can beused in the treatment of chronic and recurring pain, the treatmentthereof requiring repeated antibody administration. Thus, one particularadvantage of the anti-NGF antibodies of the invention is that theantibodies are fully human and can be administered to patients in anon-acute manner while minimizing adverse reactions commonly associatedwith human anti-mouse antibodies or other previously described non-fullyhuman antibodies from non-human species.

One skilled in the art can engineer mouse strains deficient in mouseantibody production with large fragments of the human Ig loci so thatsuch mice produce human antibodies in the absence of mouse antibodies.Large human Ig fragments may preserve the large variable gene diversityas well as the proper regulation of antibody production and expression.By exploiting the mouse cellular machinery for antibody diversificationand selection and the lack of immunological tolerance to human proteins,the reproduced human antibody repertoire in these mouse strains yieldshigh affinity antibodies against any antigen of interest, includinghuman antigens. Using the hybridoma technology, antigen-specific humanMAbs with the desired specificity may be produced and selected.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. Transfer of thehuman germ-line immunoglobulin gene array in such germ-line mutant micewill result in the production of human antibodies upon antigen challenge(see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-2555,(1993); Jakobovits et al., Nature, 362:255-258, (1993; Bruggemann etal., Year in Immun., 7:33 (1993); Nature 148:1547-1553 (1994), NatureBiotechnology 14:826 (1996); Gross, J. A., et al., Nature, 404:995-999(2000); and U.S. Pat. Nos. 5,877,397, 5,874,299, 5,814,318, 5,789,650,5,770,429, 5,661,016, 5,633,425, 5,625,126, 5,569,825, and 5,545,806(each of which is incorporated herein by reference in its entirety forall purposes)). Human antibodies can also be produced in phage displaylibraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1992); Markset al., J. Mol. Biol., 222:581 (1991)). The techniques of Cole et al.and Boerner et al. are also available for the preparation of humanmonoclonal antibodies (Cole et al., Monoclonal Antibodies and CancerTherap, Alan R. Liss, p. 77 (1985) and Boemer et al., J. Immunol.,147(l):86-95 (1991)).

Recombinant human antibodies may also be subjected to in vitromutagenesis (or, when an animal transgenic for human Ig sequences isused, in vivo somatic mutagenesis) and, thus, the amino acid sequencesof the VH and VL regions of the recombinant antibodies are sequencesthat, while derived from those related to human germline VH and VLsequences, may not naturally exist within the human antibody germlinerepertoire in vivo.

In certain embodiments, the skilled artisan can use constant regionsfrom species other than human along with the human variable region(s) insuch mice to produce chimeric antibodies.

Naturally Occurring Antibody Structure

Naturally occurring antibody structural units typically comprise atetramer. Each such tetramer typically is composed of two identicalpairs of polypeptide chains, each pair having one full-length “light”chain (typically having a molecular weight of about 25 kDa) and onefull-length “heavy” chain (typically having a molecular weight of about50-70 kDa). The amino-terminal portion of each light and heavy chaintypically includes a variable region of about 100 to 110 or more aminoacids that typically is responsible for antigen recognition. Thecarboxy-terminal portion of each chain typically defines a constantregion responsible for effector function. Human light chains aretypically classified as kappa and lambda light chains. Heavy chains aretypically classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgGhas several subclasses, including, but not limited to, IgG1, IgG2, IgG3,and IgG4. IgM has subclasses including, but not limited to, IgM1 andIgM2. IgA is similarly subdivided into subclasses including, but notlimited to, IgA1 and IgA2. Within full-length light and heavy chains,typically, the variable and constant regions are joined by a “J” regionof about 12 or more amino acids, with the heavy chain also including a“D” region of about 10 more amino acids. See, e.g., FUNDAMENTALIMMUNOLOGY, Ch. 7, 2^(nd) ed., (Paul, W., ed.), 1989, Raven Press, N.Y.(incorporated by reference in its entirety for all purposes). Thevariable regions of each light/heavy chain pair typically form theantigen-binding site.

The variable regions typically exhibit the same general structure ofrelatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. The CDRs from the two chains of each pair typically are alignedby the framework regions, which may enable binding to a specificepitope. From N-terminal to C-terminal, both light and heavy chainvariable regions typically comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain istypically in accordance with the definitions of Kabat Sequences ofProteins of Immunological Interest (1987 and 1991, National Institutesof Health, Bethesda, Md.), or Chothia & Lesk, 1987, J. Mol. Biol.196:901-917; Chothia et al., 1989, Nature 342:878-883.

Bispecific or Bifunctional Antibodies

A bispecific or bifunctional antibody typically is an artificial hybridantibody having two different heavy chain/light chain pairs and twodifferent binding sites. Bispecific antibodies may be produced by avariety of methods including, but not limited to, fusion of hybridomasor linking of F(ab′) fragments. See, e.g., Songsivilai & Lachmann, 1990,Clin. Exp. Immunol. 79: 315-321; Kostelny et al., 1992, J. Immunol.148:1547-1553.

Preparation of Antibodies

The invention provides antibodies that bind to human NGF. Theseantibodies can be produced by immunization with full-length NGF orfragments thereof. The antibodies of the invention can be polyclonal ormonoclonal, and/or may be recombinant antibodies. In preferredembodiments, antibodies of the invention are human antibodies prepared,for example, by immunization of transgenic animals capable of producinghuman antibodies (see, for example, International Patent Application,Publication W0 93/12227).

The complementarity determining regions (CDRS) of the light chain andheavy chain variable regions of anti-NGF antibodies of the invention canbe grafted to framework regions (FRs) from the same, or another,species. In certain embodiments, the CDRs of the light chain and heavychain variable regions of anti-NGF antibody may be grafted to consensushuman FRs. To create consensus human FRs, FRs from several human heavychain or light chain amino acid sequences are aligned to identify aconsensus amino acid sequence. The FRs of the anti-NGF antibody heavychain or light chain can be replaced with the FRs from a different heavychain or light chain. Rare amino acids in the FRs of the heavy and lightchains of anti-NGF antibody typically are not replaced, while the restof the FR amino acids can be replaced. Rare amino acids are specificamino acids that are in positions in which they are not usually found inFRs. The grafted variable regions from anti-NGF antibodies of theinvention can be used with a constant region that is different from theconstant region of anti-NGF antibody. Alternatively, the graftedvariable regions are part of a single chain Fv antibody. CDR grafting isdescribed, e.g., in U.S. Pat. Nos. 6,180,370, 5,693,762, 5,693,761,5,585,089, and 5,530,101, which are hereby incorporated by reference forany purpose.

Antibodies of the invention are preferably prepared using transgenicmice that have a substantial portion of the human antibody producinglocus inserted in antibody-producing cells of the mice, and that arefurther engineered to be deficient in producing endogenous, murine,antibodies. Such mice are capable of producing human immunoglobulinmolecules and antibodies and do not produce or produce substantiallyreduced amounts of murine immunoglobulin molecules and antibodies.Technologies utilized for achieving this result are disclosed in thepatents, applications, and references disclosed in the specificationherein. In preferred embodiments, the skilled worker may employ methodsas disclosed in International Patent Application Publication No. WO98/24893, which is hereby incorporated by reference for any purpose. Seealso Mendez et al., 1997, Nature Genetics 15:146-156, which is herebyincorporated by reference for any purpose.

The monoclonal antibodies (mAbs) of the invention can be produced by avariety of techniques, including conventional monoclonal antibodymethodology, e.g., the standard somatic cell hybridization technique ofKohler and Milstein (1975, Nature 256:495). Although somatic cellhybridization procedures are preferred, in principle, other techniquesfor producing monoclonal antibodies can be employed, e.g., viral oroncogenic transformation of B-lymphocytes.

The preferred animal system for preparing hybridomas is the mouse.Hybridoma production in the mouse is very well established, andimmunization protocols and techniques for isolation of immunizedsplenocytes for fusion are well known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known.

In a preferred embodiment, human monoclonal antibodies directed againstNGF can be generated using transgenic mice carrying parts of the humanimmune system rather than the mouse system. These transgenic mice,referred to herein as “HuMab” mice, contain a human immunoglobulin geneminilocus that encodes unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (Lonberg et al., 1994,Nature 368:856-859). Accordingly, the mice exhibit reduced expression ofmouse IgM or κ and in response to immunization, the introduced humanheavy chain and light chain transgenes undergo class switching andsomatic mutation to generate high affinity human IgG κ monoclonalantibodies (Lonberg et al, supra.; Lonberg and Huszar, 1995, Intern.Rev. Immunol. 13:65-93; Harding and Lonberg, 1995, Ann. N.Y. Acad. Sci.764:536-546). The preparation of HuMab mice is described in detail inTaylor et al., 1992, Nucleic Acids Res. 20:6287-6295; Chen et al., 1993,International Immunology 5:647-656; Tuaillon et al., 1994, J. Immunol.152:2912-2920; Lonberg et al., 1994, Nature 368:856-859; Lonberg, 1994,Handbook of Exp. Pharmacology 113:49-101; Taylor et al., 1994,International Immunology 6:579-591; Lonberg & Huszar, 1995, Intern. Rev.Immunol. 13:65-93; Harding & Lonberg, 1995, Ann. N.Y. Acad. Sci764:536-546; Fishwild et al., 1996, Nature Biotechnology 14:845-851, thecontents of all of which are hereby incorporated by reference in theirentirety. See further U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126;5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and5,770,429; all to Lonberg and Kay, as well as U.S. Pat. No. 5,545,807 toSurani et al.; International Patent Application Publication Nos. WO93/1227, published Jun. 24, 1993; WO 92/22646, published Dec. 23, 1992;and WO 92/03918, published Mar. 19, 1992, the disclosures of all ofwhich are hereby incorporated by reference in their entirety.Alternatively, the HCo7, HCo12, and KM transgenic mice strains describedin the Examples below can be used to generate human anti-NGF antibodies.

The present invention provides human monoclonal antibodies that arespecific for and neutralize bioactive human NGF polypeptides. Alsoprovided are antibody heavy and light chain amino acid sequences whichare highly specific for and neutralize NGF polypeptides when they arebound to them. This high specificity enables the anti-human NGF humanantibodies, and human monoclonal antibodies with like specificity, to beeffective immunotherapy for NGF associated diseases.

In one aspect, the invention provides isolated human antibodies thatbind the same or essentially the same epitope as the 4D4 antibodyprovided herein.

In one aspect, the invention provides isolated human antibodiescomprising at least one of the amino acid sequences shown in SEQ ID NOS:10, 12, 14, 16, 18, 20, 22, 24, and 79-130 that binds a NGF polypeptideepitope with high affinity and has the capacity to antagonize NGFpolypeptide activity. Preferably, these antibodies binds the same oressentially the same epitope as the 4D4 antibody provided herein.

In preferred embodiments, the isolated human antibodies bind to NGFpolypeptide with a dissociation constant (K_(D)) of 1×10⁻⁹ M or less andinhibits NGF induced survival in an in vitro neutralization assay withan IC₅₀ of 1×10⁻⁷ M or less. In more preferred embodiments, the isolatedhuman antibodies bind to NGF polypeptide with a dissociation constant(K_(D)) of 1×10⁻¹⁰ M or less and inhibits NGF induced survival in an invitro neutralization assay with an IC₅₀ of 1×10⁻⁸ M or less. In an evenmore preferred embodiment, the isolated anti-NGF human antibodies bindto human NGF polypeptide with a dissociation constant (K_(D)) of 1×10⁻¹¹M or less and inhibits NGF induced survival in an in vitro assay with anIC₅₀ of 1×10⁻⁹ M or less. Examples of anti-human NGF human antibodiesthat meet the aforementioned binding and neutralization criteria areprovided herein.

The most preferred anti-human NGF human antibody of the presentinvention is referred to herein as 4D4 and has VL and VH polypeptidesequences as shown in SEQ ID NO: 12 and SEQ ID NO: 10, respectively. Thepolynucleotide sequence encoding the VL and VH of 4D4 is shown in SEQ IDNO: 11 and SEQ ID NO: 9, respectively. The properties of the anti-humanNGF human antibodies of the present invention are specifically disclosedin the Examples. Particularly notable is the high affinity for NGFpolypeptide and high capacity to antagonize NGF polypeptide activitydemonstrated herein.

The dissociation constant (K_(D)) of an anti-human NGF human antibodycan be determined by surface plasmon resonance as generally described inExample 9. Generally, surface plasmon resonance analysis measuresreal-time binding interactions between ligand (recombinant NGFpolypeptide immobilized on a biosensor matrix) and analyte (antibodiesin solution) by surface plasmon resonance (SPR) using the BIAcore system(Pharmacia Biosensor, Piscataway, N.J.). Surface plasmon analysis canalso be performed by immobilizing the analyte (antibodies on a biosensormatrix) and presenting the ligand (recombinant V in solution). Thedissociation constant (K_(D)) of an anti-human NGF human antibody canalso be determined by using KinExA methodology. In certain embodimentsof the invention, the antibodies bind to NGF with a K_(D) of betweenapproximately 10⁻⁸ M and 10⁻¹² M. The term “K_(D)”, as used herein, isintended to refer to the dissociation constant of a particularantibody-antigen interaction. For purposes of the present inventionK_(D) was determined as shown in Example 9.

In preferred embodiments, the antibodies of the invention are of theIgG1, IgG2, IgG3, or IgG4 isotype. Preferably, the antibodies are of theIgG3 isotype. More preferably, the antibodies are of the IgG1 isotype.Most preferably, the antibodies are of the IgG2 isotype. In otherembodiments, the antibodies of the invention are of the IgM, IgA, IgE,or IgD isotype. In preferred embodiments of the invention, theantibodies comprise a human kappa light chain and a human IgG1, IgG2,IgG3, or IgG4 heavy chain. Expression of antibodies of the inventioncomprising an IgG1 or an IgG2 heavy chain constant region is describedin the Examples below. In particular embodiments, the variable regionsof the antibodies are ligated to a constant region other than theconstant region for the IgG1, IgG2, IgG3, or IgG4 isotype. In certainembodiments, the antibodies of the invention have been cloned forexpression in mammalian cells.

In certain embodiments, conservative modifications to the heavy chainsand light chains of anti-NGF antibodies (and corresponding modificationsto the encoding nucleotides) will produce anti-NGF antibodies havingfunctional and chemical characteristics similar to those of the anti-NGFantibodies disclosed herein. In contrast, substantial modifications inthe functional and/or chemical characteristics of anti-NGF antibodiesmay be accomplished by selecting substitutions in the amino acidsequence of the heavy and light chains that differ significantly intheir effect on maintaining (a) the structure of the molecular backbonein the area of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain.

For example, a “conservative amino acid substitution” may involve asubstitution of a native amino acid residue with a nonnative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position. Furthermore, any native residue inthe polypeptide may also be substituted with alanine, as has beenpreviously described for “alanine scanning mutagenesis.”

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. In certain embodiments, amino acidsubstitutions can be used to identify important residues of anti-NGFantibody, or to increase or decrease the affinity of the anti-NGFantibodies described herein.

As it is well known, minor changes in an amino acid sequence such asdeletion, addition or substitution of one, a few or even several aminoacids may lead to an allelic form of the original protein which hassubstantially identical properties. Therefore, in addition to theantibodies specifically described herein, other “substantiallyhomologous” antibodies can be readily designed and manufacturedutilizing various recombinant DNA techniques well known to those skilledin the art. In general, modifications of the genes may be readilyaccomplished by a variety of well-known techniques, such assite-directed mutagenesis. Therefore, the present invention contemplates“variant” or “mutant” anti-NGF human antibodies having substantiallysimilar characteristics to the anti-NGF human antibodies disclosedherein (See, for example, WO 00/56772, all of which is herebyincorporated herein by reference). Thus, by the term “variant” or“mutant” in reference to an anti-NGF human antibody is meant any bindingmolecule (molecule X) (i) in which the hypervariable regions CDR1, CDR2,and CDR3 of the heavy chain or the hypervariable regions CDR1, CDR2, andCDR3 of the light chain taken as a whole are at least 80% homologous,preferably at least 90% homologous, more preferably at least 95%homologous to the hypervariable regions as shown in SEQ ID NOS: 14, 18,and 22 or SEQ ID NOS: 16, 20, and 24, respectively, and (ii) wherein thevariant or mutant is capable of inhibiting the activity of human NGF tothe same extent as a reference anti-NGF human antibody having frameworkregions identical to those of molecule X.

Ordinarily, an anti-NGF human antibody variant will have light and/orheavy chain CDRs, when taken as a whole, that are at least about 80%amino acid sequence identity, preferably at least about 85% sequenceidentity, yet more preferably at least about 90% sequence identity, yetmore preferably at least about 91% sequence identity, yet morepreferably at least about 92% sequence identity, yet more preferably atleast about 93% sequence identity, yet more preferably at least about94% sequence identity, yet more preferably at least about 95% sequenceidentity, yet more preferably at least about 96% sequence identity, yetmore preferably at least about 97% sequence identity, yet morepreferably at least about 98% sequence identity, yet more preferably atleast about 99% amino acid sequence identity to the amino acid sequenceas shown in SEQ ID NOS: 14, 18, and 22 and/or SEQ ID NOS: 16, 20, and24, respectively.

More preferably, an anti-NGF human antibody variant will have a lightchain variable region, when taken as a whole, that has at least about80% amino acid sequence identity, yet more preferably at least about 81% sequence identity yet, more preferably at least about 82% sequenceidentity, yet more preferably at least about 83% sequence identity, yetmore preferably at least about 84% sequence identity, yet morepreferably at least about 85% sequence identity, yet more preferably atleast about 86% sequence identity, yet more preferably at least about87% sequence identity, yet more preferably at least about 88% sequenceidentity, yet more preferably at least about 89% sequence identity, yetmore preferably at least about 90% sequence identity, yet morepreferably at least about 91% sequence identity, yet more preferably atleast about 92% sequence identity, yet more preferably at least about93% sequence identity, yet more preferably at least about 94% sequenceidentity, yet more preferably at least about 95% sequence identity, yetmore preferably at least about 96% sequence identity, yet morepreferably at least about 97% sequence identity, yet more preferably atleast about 98% sequence identity, yet more preferably at least about99% amino acid sequence identity to the amino acid sequence as shown inSEQ ID NOS: 12, 80, 82, 84, 86, 88, 89, 90, or 91 and/or a heavy chainvariable region, when taken as a whole, that has at least about 70%amino acid sequence identity, preferably at least about 75% sequenceidentity, yet more preferably at least about 80% sequence identity, yetmore preferably at least about 81% sequence identity yet, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity, yetmore preferably at least about 99% amino acid sequence identity to theamino acid sequence as shown in SEQ ID NOS:10, 81, 83, 85, or 87.

A “variant” in reference to a polynucleotide is intended to refer to annucleic acid molecule having at least about 75% nucleic acid sequenceidentity with a polynucleotide sequence of the present invention.Ordinarily, a polynucleotide variant will have at least about 75%nucleic acid sequence identity, more preferably at least about 80%nucleic acid sequence identity, yet more preferably at least about 81%nucleic acid sequence identity, yet more preferably at least about 82%nucleic acid sequence identity, yet more preferably at least about 83%nucleic acid sequence identity, yet more preferably at least about 84%nucleic acid sequence identity, yet more preferably at least about 85%nucleic acid sequence identity, yet more preferably at least about 86%nucleic acid sequence identity, yet more preferably at least about 87%nucleic acid sequence identity, yet more preferably at least about 88%nucleic acid sequence identity, yet more preferably at least about 89%nucleic acid sequence identity, yet more preferably at least about 90%nucleic acid sequence identity, yet more preferably at least about 91%nucleic acid sequence identity, yet more preferably at least about 92%nucleic acid sequence identity, yet more preferably at least about 93%nucleic acid sequence identity, yet more preferably at least about 94%nucleic acid sequence identity, yet more preferably at least about 95%nucleic acid sequence identity, yet more preferably at least about 96%nucleic acid sequence identity, yet more preferably at least about 97%nucleic acid sequence identity, yet more preferably at least about 98%nucleic acid sequence identity, yet more preferably at least about 99%nucleic acid sequence identity with a novel nucleic acid sequencedisclosed herein.

In particular embodiments, the invention provides antibodies that have apercentage of identity to an antibody of the invention, or an antibodythat comprises a heavy chain variable region, a light chain variableregion, a CDR1, CDR2, or CDR3 region that has a percentage of identityto a heavy chain variable region, a light chain variable region, a CDR1,CDR2, or CDR3 region of the invention, as shown in Example 10 herein andFIGS. 5-10.

In certain embodiments, the invention provides an isolated humanantibody that specifically binds nerve growth factor and comprises aheavy chain and a light chain, wherein the heavy chain comprises a heavychain variable region comprising an amino acid sequence that is: atleast 70% or 75% identical to the amino acid sequence as set forth inSEQ ID NO: 10, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof, at least 70%, 80%, 85%, or 95%homologous to the amino acid sequence as set forth in SEQ ID NO: 81, oran antigen-binding or an immunologically functional immunoglobulinfragment thereof, at least 70%, 80%, 85%, or 95% identical to the aminoacid sequence as set forth in SEQ ID NO: 83, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, at least75%, 80%, or 85% identical to the amino acid sequence as set forth inSEQ ID NO: 85, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof, at least, 70%, 75%, or 80% identical tothe amino acid sequence as set forth in SEQ ID NO: 87, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof; at least 56% identical to the amino acid sequence as set forthin SEQ ID NO: 79, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof

In certain embodiments, the invention provides an isolated humanantibody that specifically binds nerve growth factor and comprises aheavy chain and a light chain, wherein the light chain comprises a lightchain variable region comprising an amino acid sequence that is: atleast 70%, 75%, 80%, or 90% identical to the amino acid sequence as setforth in SEQ ID NO: 12 or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof; at least 70%, 85%, or 90%identical to the amino acid sequence as set forth in SEQ ID NO: 80, oran antigen-binding or an immunologically functional immunoglobulinfragment thereof, at least 70%, 74%, 90%, or 94% identical to the aminoacid sequence as set forth in SEQ ID NO: 88, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, at least70%, 80%, 85%, or 87% identical to the amino acid sequence as set forthin SEQ ID NO: 89, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof, at least 70%, 85%, 90%, or 94%identical to the amino acid sequence as set forth in SEQ ID NO: 90, oran antigen-binding or an immunologically functional immunoglobulinfragment thereof, at least 70%, 85%, 90%, 95%, or 99% identical to theamino acid sequence as set forth in SEQ ID NO: 91, or an antigen-bindingor an immunologically functional immunoglobulin fragment thereof; atleast 70%, 80%, 90%, 95%, or 96% identical to the amino acid sequence asset forth in SEQ ID NO: 82, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof, at least 70%, 85%, 90%, 95%,98%, or 99% identical to the amino acid sequence as set forth in SEQ IDNO: 84, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof; or at least 70%, 85%, 90%, 95%, 98%, or99% identical to the amino acid sequence as set forth in SEQ ID NO: 86,or an antigen-binding or an immunologically functional immunoglobulinfragment thereof

In certain other embodiments, the invention provides an isolated humanantibody that specifically binds nerve growth factor and comprises ahuman heavy chain CDR1, wherein the heavy chain CDR1 is an amino acidsequence that is at least 40% or 60% identical to the amino acidsequence as set forth in SEQ ID NO: 98, SEQ ID NO: 105, SEQ ID NO: 110,or SEQ ID NO: 22, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof.

In other embodiments, the invention provides an isolated human antibodythat specifically binds nerve growth factor and comprises a human heavychain CDR2, wherein the heavy chain CDR2 is an amino acid sequence thatis: at least 70%, 82%, or 94% identical to the amino acid sequence asset forth in SEQ ID NO: 99, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof; at least 70% or 76%identical to the amino acid sequence as set forth in SEQ ID NO: 106, oran antigen-binding or an immunologically functional immunoglobulinfragment thereof, at least 59% identical to the amino acid sequence asset forth in SEQ ID NO: 18, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof, at least 70% identical tothe amino acid sequence as set forth in SEQ ID NO: 117, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof, or at least 70%, 75% or 80% identical to the amino acidsequence as set forth in SEQ ID NO: 111, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof.

In yet other embodiments, the invention provides an isolated humanantibody that specifically binds nerve growth factor and comprises ahuman light chain CDR1, wherein the CDR1 is an amino acid sequence thatis: at least 70% or 80% identical to the amino acid sequence as setforth in SEQ ID NO: 101, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof; at least 70%, 75%, 80% or90% identical to the amino acid sequence as set forth in SEQ ID NO: 95,or an antigen-binding or an immunologically functional immunoglobulinfragment thereof, at least 75%, 80%, or 90% identical to the amino acidsequence as set forth in SEQ ID NO: 119, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, at least75%, 80%, or 90% identical to the amino acid sequence as set forth inSEQ ID NO: 122, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof, at least 80% identical to the aminoacid sequence as set forth in SEQ ID NO: 125, or an antigen-binding oran immunologically functional immunoglobulin fragment thereof, at least75%, 80%, or 90% identical to the amino acid sequence as set forth inSEQ ID NO: 24, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof, at least 70% or 80% identical to theamino acid sequence as set forth in SEQ ID NO: 107, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof; or at least 70% or 80% identical to the amino acid sequence asset forth in SEQ ID NO: 113, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof.

In additional embodiments, the invention provides an isolated humanantibody that specifically binds nerve growth factor and comprises ahuman light chain CDR2, wherein the CDR2 is an amino acid sequence thatis: at least 70% or 85% identical to the amino acid sequence as setforth in SEQ ID NO: 102, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof, at least 70% identical tothe amino acid sequence as set forth in SEQ ID NO: 96, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof, at least 70% identical to the amino acid sequence as set forthin SEQ ID NO: 120, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof, at least 70% identical tothe amino acid sequence as set forth in SEQ ID NO: 123, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof, at least 70% or 85% identical to the amino acid sequence as setforth in SEQ ID NO: 126, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof, at least 70% or 85%identical to the amino acid sequence as set forth in SEQ ID NO: 129, oran antigen-binding or an immunologically functional immunoglobulinfragment thereof, at least 70% identical to the amino acid sequence asset forth in SEQ ID NO: 20, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof, at least 70% or 85%identical to the amino acid sequence as set forth in SEQ ID NO: 108, oran antigen-binding or an immunologically functional immunoglobulinfragment thereof, at least 70% identical to the amino acid sequence asset forth in SEQ ID NO: 133, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof, or at least 70% or 85%identical to the amino acid sequence as set forth in SEQ ID NO: 114, oran antigen-binding or an immunologically functional immunoglobulinfragment thereof.

In other embodiments, the invention provides an isolated human antibodythat specifically binds nerve growth factor and comprises a human lightchain CDR3, wherein the CDR3 is an amino acid sequence that is: at least70% or 85% identical to the amino acid sequence as set forth in SEQ IDNO: 103, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof, at least 70% or 85% identical to theamino acid sequence as set forth in SEQ ID NO: 97, or an antigen-bindingor an immunologically functional immunoglobulin fragment thereof, atleast 70% or 78% identical to the amino acid sequence as set forth inSEQ ID NO: 121, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof, at least 70% or 78% identical to theamino acid sequence as set forth in SEQ ID NO: 127, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof, at least 70% or 78% identical to the amino acid sequence as setforth in SEQ ID NO: 130, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof, at least 70% or 78%identical to the amino acid sequence as set forth in SEQ ID NO: 16, oran antigen-binding or an immunologically functional immunoglobulinfragment thereof, at least 70% or 85% identical to the amino acidsequence as set forth in SEQ ID NO: 109, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, at least 78%identical to the amino acid sequence as set forth in SEQ ID NO: 134, oran antigen-binding or an immunologically functional immunoglobulinfragment thereof; or at least 85% identical to the amino acid sequenceas set forth in SEQ ID NO: 115, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof

The sequences of the 4D4 antibody heavy chain and light chain variableregions are shown in SEQ ID NOS: 10 and 12, respectively. However, manyof the potential CDR-contact residues are amenable to substitution byother amino acids and still allow the antibody to retain substantialaffinity for the antigen. Likewise, many of the framework residues notin contact with the CDRs in the heavy and light chains can accommodatesubstitutions of amino acids from the corresponding positions from otherhuman antibodies, by human consensus amino acids, or from other mouseantibodies, without significant loss of the affinity ornon-immunogenicity of the human antibody. Selection of variousalternative amino acids may be used to produce versions of the disclosedanti-NGF antibodies and fragments thereof that have varying combinationsof affinity, specificity, non-immunogenicity, ease of manufacture, andother desirable properties.

In alternative embodiments, antibodies of the invention can be expressedin cell lines other than hybridoma cell lines. In these embodiments,sequences encoding particular antibodies can be used for transformationof a suitable mammalian host cell. According to these embodiments,transformation can be achieved using any known method for introducingpolynucleotides into a host cell, including, for example packaging thepolynucleotide in a virus (or into a viral vector) and transducing ahost cell with the virus (or vector) or by transfection procedures knownin the art, as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040,4,740,461, and 4,959,455 (all of which are hereby incorporated herein byreference for any purpose). Generally, the transformation procedure usedmay depend upon the host to be transformed. Methods for introducingheterologous polynucleotides into mammalian cells are well known in theart and include, but are not limited to, dextran-mediated transfection,calcium phosphate precipitation, polybrene mediated transfection,protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

A nucleic acid molecule encoding the amino acid sequence of a heavychain constant region, a heavy chain variable region, a light chainconstant region, or a light chain variable region of an NGF antibody ofthe invention is inserted into an appropriate expression vector usingstandard ligation techniques. In a preferred embodiment, the anti-NGFantibody heavy chain or light chain constant region is appended to theC-terminus of the appropriate variable region and is ligated into anexpression vector. The vector is typically selected to be functional inthe particular host cell employed (i.e., the vector is compatible withthe host cell machinery such that amplification of the gene and/orexpression of the gene can occur). For a review of expression vectors,see METH. ENZ. 185 (Goeddel, ed.), 1990, Academic Press.

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” in certain embodiments will typically includeone or more of the following nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these sequences is discussed below.

Optionally, the vector may contain a “tag”-encoding sequence, i. e., anoligonucleotide molecule located at the 5′ or 3′ end of the anti-NGFantibody polypeptide coding sequence; the oligonucleotide sequenceencodes polyHis (such as hexaHis), or another “tag” such as FLAG, HA(hemaglutinin influenza virus), or myc for which commercially availableantibodies exist. This tag is typically fused to the polypeptide uponexpression of the polypeptide, and can serve as a means for affinitypurification or detection of the NGF antibody from the host cell.Affinity purification can be accomplished, for example, by columnchromatography using antibodies against the tag as an affinity matrix.Optionally, the tag can subsequently be removed from the purifiedanti-NGF antibody polypeptide by various means such as using certainpeptidases for cleavage.

Flanking sequences may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), synthetic or native. Assuch, the source of a flanking sequence may be any prokaryotic oreukaryotic organism, any vertebrate or invertebrate organism, or anyplant, provided that the flanking sequence is functional in, and can beactivated by, the host cell machinery.

Flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein will have been previously identified bymapping and/or by restriction endonuclease digestion and can thus beisolated from the proper tissue source using the appropriate restrictionendonucleases. In some cases, the full nucleotide sequence of a flankingsequence may be known. Here, the flanking sequence may be synthesizedusing the methods described herein for nucleic acid synthesis orcloning.

Whether all or only a portion of the flanking sequence is known, it maybe obtained using polymerase chain reaction (PCR) and/or by screening agenomic library with a suitable probe such as an oligonucleotide and/orflanking sequence fragment from the same or another species. Where theflanking sequence is not known, a fragment of DNA containing a flankingsequence may be isolated from a larger piece of DNA that may contain,for example, a coding sequence or even another gene or genes. Isolationmay be accomplished by restriction endonuclease digestion to produce theproper DNA fragment followed by isolation using agarose gelpurification, Qiagen® column chromatography (Chatsworth, Calif.), orother methods known to the skilled artisan. The selection of suitableenzymes to accomplish this purpose will be readily apparent to one ofordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. If the vector of choice doesnot contain an origin of replication site, one may be chemicallysynthesized based on a known sequence, and ligated into the vector. Forexample, the origin of replication from the plasmid pBR322 (New EnglandBiolabs, Beverly, Mass.) is suitable for most gram-negative bacteria,and various viral origins (e.g., SV40, polyoma, adenovirus, vesicularstomatitus virus (VSV), or papillomaviruses such as HPV or BPV) areuseful for cloning vectors in mammalian cells. Generally, the origin ofreplication component is not needed for mammalian expression vectors(for example, the SV40 origin is often used only because it alsocontains the virus early promoter).

A transcription termination sequence is typically located 3′ to the endof a polypeptide coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly-T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene encodes a protein necessary for the survivaland growth of a host cell grown in a selective culture medium. Typicalselection marker genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, tetracycline, orkanamycin for prokaryotic host cells; (b) complement auxotrophicdeficiencies of the cell; or (c) supply critical nutrients not availablefrom complex or defined media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. Advantageously, a neomycin resistance genemay also be used for selection in both prokaryotic and eukaryotic hostcells.

Other selectable genes may be used to amplify the gene that will beexpressed. Amplification is the process wherein genes that are requiredfor production of a protein critical for growth or cell survival arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and promoterless thymidinekinase genes. Mammalian cell transformants are placed under selectionpressure wherein only the transformants are uniquely adapted to surviveby virtue of the selectable gene present in the vector. Selectionpressure is imposed by culturing the transformed cells under conditionsin which the concentration of selection agent in the medium issuccessively increased, thereby leading to the amplification of both theselectable gene and the DNA that encodes another gene, such as anantibody that binds to NGF polypeptide. As a result, increasedquantities of a polypeptide such as an anti-NGF antibody are synthesizedfrom the amplified DNA.

A ribosome-binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the polypeptide to beexpressed.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various pre- orprosequences to improve glycosylation or yield. For example, one mayalter the peptidase cleavage site of a particular signal peptide, or addpro-sequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein) one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid residues found inthe peptidase cleavage site, attached to the amino-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired polypeptide, if the enzyme cutsat such area within the mature polypeptide.

Expression and cloning vectors of the invention will typically contain apromoter that is recognized by the host organism and operably linked tothe molecule encoding the anti-NGF antibody. Promoters are untranscribedsequences located upstream (i.e., 5′) to the start codon of a structuralgene (generally within about 100 to 1000 bp) that control transcriptionof the structural gene. Promoters are conventionally grouped into one oftwo classes: inducible promoters and constitutive promoters. Induciblepromoters initiate increased levels of transcription from DNA undertheir control in response to some change in culture conditions, such asthe presence or absence of a nutrient or a change in temperature.Constitutive promoters, on the other hand, uniformly transcribe gene towhich they are operably linked, that is, with little or no control overgene expression. A large number of promoters, recognized by a variety ofpotential host cells, are well known. A suitable promoter is operablylinked to the DNA encoding heavy chain or light chain comprising ananti-NGF antibody of the invention by removing the promoter from thesource DNA by restriction enzyme digestion and inserting the desiredpromoter sequence into the vector.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retroviruses, hepatitis-B virus and most preferablySimian Virus 40 (SV40). Other suitable mammalian promoters includeheterologous mammalian promoters, for example, heat-shock promoters andthe actin promoter.

Additional promoters which may be of interest include, but are notlimited to: SV40 early promoter (Bernoist and Chambon, 1981, Nature290:304-10); CMV promoter (Thomsen et al., 1984, Proc. Natl. Acad. USA81:659-663); the promoter contained in the 3′ long terminal repeat ofRous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-97); herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:1444-45); promoter and regulatory sequences from themetallothionine gene (Brinster et al., 1982, Nature 296:39-42); andprokaryotic promoters such as the beta-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A.,75:3727-31); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad.Sci. U.S.A., 80:21-25). Also of interest are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion that is active in pancreatic acinar cells (Swift et al., 1984,Cell 38:639-46; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant.Biol. 50:399-409 (1986); MacDonald, 1987, Hepatology 7:425-515); theinsulin gene control region that is active in pancreatic beta cells(Hanahan, 1985, Nature 315:115-22); the immunoglobulin gene controlregion that is active in lymphoid cells (Grosschedl et al., 1984, Cell38:647-58; Adames et al., 1985, Nature 318:533-38; Alexander et al.,1987, Mol. Cell Biol., 7:1436-44); the mouse mammary tumor virus controlregion that is active in testicular, breast, lymphoid and mast cells(Leder et al., 1986, Cell 45:485-95); the albumin gene control regionthat is active in liver (Pinkert et al., 1987, Genes and Devel.1:268-76); the alpha-feto-protein gene control region that is active inliver (Kirumlauf et al., 1985, Mol. Cell. Biol., 5:1639-48; Hammer etal., 1987, Science 235:53-58); the alpha 1-antitrypsin gene controlregion that is active in liver (Kelsey et al., 1987, Genes and Devel.1:161-71); the beta-globin gene control region that is active in myeloidcells (Mogram et al., 1985, Nature 315:338-40; Kollias et al., 1986,Cell 46:89-94); the myelin basic protein gene control region that isactive in oligodendrocyte cells in the brain (Readhead et al., 1987,Cell 48:703-12); the myosin light chain-2 gene control region that isactive in skeletal muscle (Sani, 1985, Nature 314:283-86); and thegonadotropic releasing hormone gene control region that is active in thehypothalamus (Mason et al., 1986, Science 234:1372-78).

An enhancer sequence may be inserted into the vector to increasetranscription of DNA encoding light chain or heavy chain comprising ananti-NGF antibody of the invention by higher eukaryotes. Enhancers arecis-acting elements of DNA, usually about 10-300 bp in length, that acton the promoter to increase transcription. Enhancers are relativelyorientation and position independent, having been found at positionsboth 5′ and 3′ to the transcription unit. Several enhancer sequencesavailable from mammalian genes are known (e.g., globin, elastase,albumin, alpha-feto-protein and insulin). Typically, however, anenhancer from a virus is used. The SV40 enhancer, the cytomegalovirusearly promoter enhancer, the polyoma enhancer, and adenovirus enhancersknown in the art are exemplary enhancing elements for the activation ofeukaryotic promoters. While an enhancer may be positioned in the vectoreither 5′ or 3′ to a coding sequence, it is typically located at a site5′ from the promoter.

Expression vectors of the invention may be constructed from a startingvector such as a commercially available vector. Such vectors may or maynot contain all of the desired flanking sequences. Where one or more ofthe flanking sequences described herein are not already present in thevector, they may be individually obtained and ligated into the vector.Methods used for obtaining each of the flanking sequences are well knownto one skilled in the art.

After the vector has been constructed and a nucleic acid moleculeencoding light chain, a heavy chain, or a light chain and a heavy chaincomprising an anti-NGF antibody has been inserted into the proper siteof the vector, the completed vector may be inserted into a suitable hostcell for amplification and/or polypeptide expression. The transformationof an expression vector for an anti-NGF antibody into a selected hostcell may be accomplished by well known methods including transfection,infection, calcium phosphate co-precipitation, electroporation,microinjection, lipofection, DEAE-dextran mediated transfection, orother known techniques. The method selected will in part be a functionof the type of host cell to be used. These methods and other suitablemethods are well known to the skilled artisan, and are set forth, forexample, in Sambrook et al., supra.

A host cell, when cultured under appropriate conditions, synthesizes ananti-NGF antibody that can subsequently be collected from the culturemedium (if the host cell secretes it into the medium) or directly fromthe host cell producing it (if it is not secreted). The selection of anappropriate host cell will depend upon various factors, such as desiredexpression levels, polypeptide modifications that are desirable ornecessary for activity (such as glycosylation or phosphorylation) andease of folding into a biologically active molecule

Mammalian cell lines available as hosts for expression are well known inthe art and include, but are not limited to, immortalized cell linesavailable from the American Type Culture Collection (ATCC), includingbut not limited to Chinese hamster ovary (CHO) cells, HeLa cells, babyhamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), and a number of othercell lines. In certain embodiments, cell lines may be selected throughdetermining which cell lines have high expression levels andconstitutively produce antibodies with NGF binding properties. Inanother embodiment, a cell line from the B cell lineage that does notmake its own antibody but has a capacity to make and secrete aheterologous antibody can be selected.

Antibodies of the invention are useful for detecting NGF in biologicalsamples and identification of cells or tissues that produce NGF protein.Antibodies of the invention that specifically bind to NGF may be usefulin treatment of NGF mediated diseases. Said antibodies can be used inbinding assays to detect NGF and to inhibit NGF from forming a complexwith NGF receptors. Said antibodies that bind to NGF and blockinteraction with other binding compounds may have therapeutic use inmodulating NGF mediated diseases. In preferred embodiments, antibodiesto NGF may block NGF binding to its receptor, which may result indisruption of the NGF induced signal transduction cascade.

The present invention also relates to the use of one or more of theantibodies of the present invention in the manufacture of a medicamentfor the treatment of a painful disorder or condition caused by increasedexpression of NGF or increased sensitivity to NGF in a patient such asany one of disorders or conditions disclosed herein.

In preferred embodiments, the invention provides pharmaceuticalcompositions comprising a therapeutically effective amount of one or aplurality of the antibodies of the invention together with apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative and/or adjuvant. Preferably, acceptable formulationmaterials are nontoxic to recipients at the dosages and concentrationsemployed. In preferred embodiments, pharmaceutical compositionscomprising a therapeutically effective amount of anti-NGF antibodies areprovided.

In certain embodiments, acceptable formulation materials preferably arenontoxic to recipients at the dosages and concentrations employed.

In certain embodiments, the pharmaceutical composition may containformulation materials for modifying, maintaining or preserving, forexample, the pH, osmolarity, viscosity, clarity, color, isotonicity,odor, sterility, stability, rate of dissolution or release, adsorptionor penetration of the composition. In such embodiments, suitableformulation materials include, but are not limited to, amino acids (suchas glycine, glutamine, asparagine, arginine or lysine); antimicrobials;antioxidants (such as ascorbic acid, sodium sulfite or sodiumhydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl,citrates, phosphates or other organic acids); bulking agents (such asmannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants. SeeREMINGTON'S PHARMACEUTICAL SCIENCES, 18^(th) Edition, (A. R. Gennaro,ed.), 1990, Mack Publishing Company.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theantibodies of the invention.

In certain embodiments, the primary vehicle or carrier in apharmaceutical composition may be either aqueous or non-aqueous innature. For example, a suitable vehicle or carrier may be water forinjection, physiological saline solution or artificial cerebrospinalfluid, possibly supplemented with other materials common in compositionsfor parenteral administration. Neutral buffered saline or saline mixedwith serum albumin are further exemplary vehicles. In preferredembodiments, pharmaceutical compositions of the present inventioncomprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH4.0-5.5, and may further include sorbitol, sucrose, Tween-20 and/or asuitable substitute therefor. In certain embodiments of the invention,anti-NGF antibody compositions may be prepared for storage by mixing theselected composition having the desired degree of purity with optionalformulation agents (REMINGTON'S PHARMACEUTICAL SCIENCES, supra) in theform of a lyophilized cake or an aqueous solution. Further, in certainembodiments, the anti-NGF antibody product may be formulated as alyophilizate using appropriate excipients such as sucrose.

The pharmaceutical compositions of the invention can be selected forparenteral delivery. Alternatively, the compositions may be selected forinhalation or for delivery through the digestive tract, such as orally.Preparation of such pharmaceutically acceptable compositions is withinthe skill of the art.

The formulation components are present preferably in concentrations thatare acceptable to the site of administration. In certain embodiments,buffers are used to maintain the composition at physiological pH or at aslightly lower pH, typically within a pH range of from about 5 to about8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be provided in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired anti-NGF antibody in a pharmaceutically acceptable vehicle. Aparticularly suitable vehicle for parenteral injection is steriledistilled water in which the anti-NGF antibody is formulated as asterile, isotonic solution, properly preserved. In certain embodiments,the preparation can involve the formulation of the desired molecule withan agent, such as injectable microspheres, bio-erodible particles,polymeric compounds (such as polylactic acid or polyglycolic acid),beads or liposomes, that may provide controlled or sustained release ofthe product which can be delivered via depot injection. In certainembodiments, hyaluronic acid may also be used, having the effect ofpromoting sustained duration in the circulation. In certain embodiments,implantable drug delivery devices may be used to introduce the desiredantibody molecule.

Pharmaceutical compositions of the invention can be formulated forinhalation. In these embodiments, anti-NGF antibodies are advantageouslyformulated as a dry, inhalable powder. In preferred embodiments,anti-NGF antibody inhalation solutions may also be formulated with apropellant for aerosol delivery. In certain embodiments, solutions maybe nebulized. Pulmonary administration and formulation methods thereforeare further described in International Patent Application No.PCT/US94/001875, which is incorporated by reference and describespulmonary delivery of chemically modified proteins.

It is also contemplated that formulations can be administered orally.Anti-NGF antibodies that are administered in this fashion can beformulated with or without carriers customarily used in the compoundingof solid dosage forms such as tablets and capsules. In certainembodiments, a capsule may be designed to release the active portion ofthe formulation at the point in the gastrointestinal tract whenbioavailability is maximized and pre-systemic degradation is minimized.Additional agents can be included to facilitate absorption of theanti-NGF antibody. Diluents, flavorings, low melting point waxes,vegetable oils, lubricants, suspending agents, tablet disintegratingagents, and binders may also be employed.

A pharmaceutical composition of the invention is preferably provided tocomprise an effective quantity of one or a plurality of anti-NGFantibodies in a mixture with non-toxic excipients that are suitable forthe manufacture of tablets. By dissolving the tablets in sterile water,or another appropriate vehicle, solutions may be prepared in unit-doseform. Suitable excipients include, but are not limited to, inertdiluents, such as calcium carbonate, sodium carbonate or bicarbonate,lactose, or calcium phosphate; or binding agents, such as starch,gelatin, or acacia; or lubricating agents such as magnesium stearate,stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving anti-NGF antibodies insustained- or controlled-delivery formulations. Techniques forformulating a variety of other sustained- or controlled-delivery means,such as liposome carriers, bio-erodible microparticles or porous beadsand depot injections, are also known to those skilled in the art. See,for example, International Patent Application No. PCT/US93/00829, whichis incorporated by reference and describes controlled release of porouspolymeric microparticles for delivery of pharmaceutical compositions.Sustained-release preparations may include semipermeable polymermatrices in the form of shaped articles, e.g. films, or microcapsules.Sustained release matrices may include polyesters, hydrogels,polylactides (as disclosed in U.S. Pat. No. 3,773,919 and EuropeanPatent Application Publication No. EP 058481, each of which isincorporated by reference), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22:547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater.Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinylacetate (Langer et al., supra) or poly-D(-)-3-hydroxybutyric acid(European Patent Application Publication No. EP 133,988). Sustainedrelease compositions may also include liposomes that can be prepared byany of several methods known in the art. See e.g., Eppstein et al.,1985, Proc. Natl. Acad. Sci. USA 82:3688-3692; European PatentApplication Publication Nos. EP 036,676; EP 088,046 and EP 143,949,incorporated by reference.

Pharmaceutical compositions used for in vivo administration aretypically provided as sterile preparations. Sterilization can beaccomplished by filtration through sterile filtration membranes. Whenthe composition is lyophilized, sterilization using this method may beconducted either prior to or following lyophilization andreconstitution. Compositions for parenteral administration can be storedin lyophilized form or in a solution. Parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or as a dehydrated or lyophilized powder. Such formulations may bestored either in a ready-to-use form or in a form (e.g., lyophilized)that is reconstituted prior to administration.

The invention also provides kits for producing a single-doseadministration unit. The kits of the invention may each contain both afirst container having a dried protein and a second container having anaqueous formulation. In certain embodiments of this invention, kitscontaining single and multi-chambered pre-filled syringes (e.g., liquidsyringes and lyosyringes) are provided.

The effective amount of an anti-NGF antibody-containing pharmaceuticalcomposition to be employed therapeutically will depend, for example,upon the therapeutic context and objectives. One skilled in the art willappreciate that the appropriate dosage levels for treatment will varydepending, in part, upon the molecule delivered, the indication forwhich the anti-NGF antibody is being used, the route of administration,and the size (body weight, body surface or organ size) and/or condition(the age and general health) of the patient. In certain embodiments, theclinician may titer the dosage and modify the route of administration toobtain the optimal therapeutic effect. A typical dosage may range fromabout 0.1 μg/kg to up to about 30 mg/kg or more, depending on thefactors mentioned above. In preferred embodiments, the dosage may rangefrom 0.1 μg/kg up to about 30 mg/kg; more preferably from 1 μg/kg up toabout 30 mg/kg; or even more preferably from 5 μg/kg up to about 30mg/kg.

In certain embodiments, the compositions can be administered bysubcutaneous injection. As noted above the dosage amount can bedetermined by a clinician, however in certain embodiments thepharmaceutically effective amount of an NGF antibody in the compositionto be administered by subcutaneous injection is from about 0.1 μg/kg upto about 30 mg/kg. In certain embodiments, the pharmaceuticallyeffective amount an NGF antibody is from about 3 mg to about 30 mg persubcutaneous injection. In certain embodiments the administrationcomprises multiple subcutaneous injections. In yet other embodiments,the administration comprises a single subcutaneous injection.

Dosing frequency will depend upon the pharmacokinetic parameters of theparticular anti-NGF antibody in the formulation used. Typically, aclinician administers the composition until a dosage is reached thatachieves the desired effect. The composition may therefore beadministered as a single dose, or as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. Appropriate dosages may be ascertained through use ofappropriate dose-response data. In certain embodiments, the antibodiesof the invention can be administered to patients throughout an extendedtime period. Chronic administration of an antibody of the inventionminimizes the adverse immune or allergic response commonly associatedwith antibodies that are raised against a human antigen in a non-humananimal, for example, a non-fully human antibody produced in a non-humanspecies.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g. orally, through injection byintravenous, intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, or intralesional routes; by sustained release systems or byimplantation devices. In certain embodiments, the compositions may beadministered by bolus injection or continuously by infusion, or byimplantation device.

The composition also may be administered locally via implantation of amembrane, sponge or another appropriate material onto which the desiredmolecule has been absorbed or encapsulated. In certain embodiments,where an implantation device is used, the device may be implanted intoany suitable tissue or organ, and delivery of the desired molecule maybe via diffusion, timed-release bolus, or continuous administration.

In certain embodiments, the invention relates to a method of treating acondition caused by increased expression of nerve growth factor (NGF) orincreased sensitivity to NGF comprising administering to a patientorally, through injection by intravenous, intraperitoneal, intracerebral(intra-parenchymal), intracerebroventricular, intramuscular,intra-ocular, intraarterial, intraportal, intralesional or subcutaneousroutes, by sustained release systems or by implantation devices apharmaceutically effective amount of an NGF antibody., wherein thecondition is acute pain, dental pain, pain from trauma, surgical pain,pain resulting from amputation or abscess, causalgia, demyelinatingdiseases, trigeminal neuralgia, cancer, chronic alcoholism, stroke,thalamic pain syndrome, diabetes, acquired immune deficiency syndrome(“AIDS”), toxins, chemotherapy, general headache, migraine, clusterheadache, mixed-vascular or non-vascular syndromes, tension headache,general inflammation, arthritis, rheumatic diseases, lupus,osteoarthritis, fibromyalgia, inflammatory bowel disorders, irritablebowel syndrome, inflammatory eye disorders, inflammatory or unstablebladder disorders, psoriasis, skin complaints with inflammatorycomponents, sunburn, carditis, dermatitis, myositis, neuritis, collagenvascular diseases, chronic inflammatory conditions, inflammatory painand associated hyperalgesia and allodynia, neuropathic pain andassociated hyperalgesia or allodynia, diabetic neuropathy pain,causalgia, sympathetically maintained pain, deafferentation syndromes,asthma, epithelial tissue damage or dysfunction, herpes simplex,disturbances of visceral motility at respiratory, genitourinary,gastrointestinal or vascular regions, wounds, burns, allergic skinreactions, pruritis, vitiligo, general gastrointestinal disorders,colitis, gastric ulceration, duodenal ulcers, vasomotor or allergicrhinitis, or bronchial disorders, dysmenorrhoea, dyspepsia,gastroesophageal reflux, pancreatitis, or visceralgia.

In certain embodiments, the methods comprise a pharmaceuticallyeffective amount of an NGF antibody and are useful for treating orpreventing osteoarthritis knee pain. In certain embodiments thepharmaceutically effective amount an NGF antibody is from about 3 mg toabout 30 mg per subcutaneous injection. In certain embodiments theadministration comprises multiple subcutaneous injections. Inembodiments, the administration comprises a single subcutaneousinjection. In certain embodiments, the compositions and methods of theinvention comprise an NGF antibody comprising a light chain comprisingSEQ ID NO. 44. In certain embodiments, the NGF antibody comprises aheavy chain comprising SEQ ID. NO. 40. In further embodiments, the NGFantibody comprises a light chain comprising SEQ ID NO. 44, and a heavychain comprising SEQ ID. NO. 40.

Accordingly, in accordance with the above description of the invention,in various aspects the invention relates to methods and compositionscomprising an NGF antibody comprising a light chain comprising SEQ IDNO: 44 and a heavy chain comprising SEQ ID NO: 40, wherein the heavychain and light chain of the antibody are connected by a flexible linkerto form a single chain antibody. In some embodiments of this aspect, theNGF antibody comprises a single-chain Fv antibody, a Fab′ antibody, a(Fab′)₂ antibody, a fully human antibody, and/or a humanized antibody.In some embodiments of this aspect the NGF antibody inhibits NGFsignaling.

In certain embodiments of this aspect, the NGF antibody dissociates froma human NGF polypeptide with a K_(D) of about 1×10⁻⁹ or less, about1×10⁻¹⁰ or less, or about 1×10⁻¹¹ or less. In certain embodiments ofthis aspect, the NGF antibody neutralizes human NGF bioactivity in astandard in vitro assay with an IC₅₀ of about 1×10⁻⁸ or less, about1×10⁻⁹ or less, or about 0.2×10⁻⁹ or less. In certain embodiments, theNGF antibody dissociates from a human NGF polypeptide with theabove-mentioned K_(D) value(s) and neutralizes human NGF bioactivity ina standard in vitro assay with the above-mentioned IC₅₀ values.

It also may be desirable to use anti-NGF antibody pharmaceuticalcompositions according to the invention ex vivo. In such instances,cells, tissues or organs that have been removed from the patient areexposed to anti-NGF antibody pharmaceutical compositions after which thecells, tissues and/or organs are subsequently implanted back into thepatient.

In particular, anti-NGF antibodies can be delivered by implantingcertain cells that have been genetically engineered, using methods suchas those described herein, to express and secrete the polypeptide. Incertain embodiments, such cells may be animal or human cells, and may beautologous, heterologous, or xenogeneic. In certain embodiments, thecells may be immortalized. In other embodiments, in order to decreasethe chance of an immunological response, the cells may be encapsulatedto avoid infiltration of surrounding tissues. In further embodiments,the encapsulation materials are typically biocompatible, semi-permeablepolymeric enclosures or membranes that allow the release of the proteinproduct(s) but prevent the destruction of the cells by the patient'simmune system or by other detrimental factors from the surroundingtissues.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting the invention.

Example 1

Generation of Human NGF Protein from E. coli CellsCloning of rHu-NGF (1-120)

The nucleotide sequence encoding human NGF was amplified from cDNA usingthe oligonucleotide primers with sequences as shown in SEQ ID NO:27 andSEQ ID NO:28 and standard PCR technology. The 5′ primer creates an NdeIrestriction site and methionine initiation codon immediately precedingcodon 1 (serine) of the mature sequence. The 3′ primer creates a BamHIrestriction site immediately following the termination codon. Theresulting PCR product was gel purified, digested with restrictionendonucleases NdeI and BamHI, and then ligated into the vector pCFM1656,also digested with NdeI and BamHI. Ligated DNA was transformed intocompetent host cells of E. coli strain 657. Clones were screened for theability to produce the recombinant protein product and to possess aplasmid having the correct nucleotide sequence (i.e., SEQ ID NO:29). Theamino acid sequence of the recombinant human NGF 1-120 is shown as SEQID NO:30:

The expression vector pCFM1656 (ATCC #69576) was derived from theexpression vector system described in U.S. Pat. No. 4,710,473. ThepCFM1656 plasmid can be derived from the described pCFM836 plasmid (U.S.Pat. No. 4,710,473) by: (a) destroying the two endogenous NdeIrestriction sites by end filling with T4 polymerase enzyme followed byblunt end ligation; (b) replacing the DNA sequence between the uniqueAatII and CaI restriction sites containing the synthetic P_(L) promoterwith a similar fragment obtained from pCFM636 (U.S. Pat. No. 4,710,473)containing the PL promoter and then (c) substituting the small DNAsequence between the unique ClaI and KpnI restriction sites witholigonucleotide resulting from annealling two probes have nucleotidesequences as shown in SEQ ID NO: 31 and SEQ ID NO:32.

The E. coli K12 host strain (Amgen strain 657) is a derivative of E.coli W1485 (a K12 strain), obtained from the E. coli Genetic StockCenter, Yale University, New Haven, Conn. (CGSC strain 6159).

Expression of rHu-NGF(1-120)

E. coli cells containing the NGF expression construct (as describedabove) were fermented in rich medium in fed-batch mode. Cells were grownat 30° C. to an OD at 600 nm of 49, and then induced by temperatureshift to 42° C. Cells were harvested by centrifugation at four hourspost induction. Final OD was 75. Expression yield was determined to beapproximately 0.15 g/L.

Refolding and Purification of rHu-NGF(1-120)

Cell paste was lysed in a Microfluidizer, centrifuged at 10,000×g for 30minutes, the pellet was washed with 1% deoxycholic acid, centrifuged asabove, and the resulting pellet was then washed with cold water andre-centrifuged. The resulting pellet (WIBs—washed inclusion bodies) wasresuspended in denaturant, 8M guanidine HCl, 50 mM Tris pH 8.5,containing 10 mM DTT, and solubilized at room temperature for 1 hour,centrifuged at 10,000×g for 30 minutes, and the supernatent wascarefully decanted and then diluted 25-fold into an aqueous buffercontaining a redox couple at 4° C., for 5 days. The resulting refold wasthen titrated to pH 3.0, filtered through a 0.45 uM filter. The refoldwas purified using a Sp-Sepharose fast flow column using a standard NaClgradient. The pool from the cation exchange column was subsequentlyconcentrated and aliquots were frozen −80° C. The purity of the proteinwas assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) andanalyzed by Coomassie blue stain. The purified protein was greater than90% main band by this method.

Example 2

Production of Human Monoclonal Antibodies against Nerve Growth Factor(NGF) Transgenic HuMab and KM Mice

Fully human monoclonal antibodies to NGF were prepared using HCo7,HCo12, HCo7+HCo12, and KM strains of transgenic mice, each of whichexpresses human antibody genes. In each of these mouse strains, theendogenous mouse kappa light chain gene has been homozygously disruptedas described in Chen et al. (1993, EMBO J. 12:811-820), and theendogenous mouse heavy chain gene has been homozygously disrupted asdescribed in Example 1 of International Patent Application PublicationNo. WO 01/09187 (incorporated by reference). Each of these mouse strainscarries a human kappa light chain transgene, KCo5, as described inFishwild et al. (1996, Nature Biotechnology 14:845-851). The HCo7 straincarries the HCo7 human heavy chain transgene as described in U.S. Pat.Nos. 5,545,806, 5,625,825, and 5,545,807 (incorporated by reference).The HCo12 strain carries the HCo12 human heavy chain transgene asdescribed in Example 2 of International Patent Application PublicationNo. WO 01/09187 (incorporated by reference). The HCo7+HCo12 straincarries both the HCo7 and the HCo12 heavy chain transgenes and ishemizygous for each transgene. The KM mice comprises the SC20 heavychain transgene as described in Tomizuka et al. (1997, Nature Genet. 16,133-143 and 2000, Proc. Natl. Acad. Sci, 97, 722-727). This transgene isnot integrated into a mouse chromosome, but is instead propagated as anindependent chromosome fragment. The fragment includes approximately 15MB of human chromosome 14. It contains the entire human heavy chainlocus including all VH, D and JH gene segments and all heavy chainconstant region isotypes. All of these strains are referred to herein asHuMab mice.

HuMab Immunizations:

To generate fully human monoclonal antibodies to NGF, HuMab mice wereimmunized with purified recombinant NGF derived from E. coli cells asantigen (Example 1). General immunization schemes for HuMab mice aredescribed in Lonberg et al. (1994, Nature 368:856-859; Fishwild et al.,supra., and International Patent Application Publication No. WO98/24884, the teachings of each of which are incorporated by reference).Mice were 6-16 weeks of age upon the first infusion of antigen. Apurified recombinant preparation (25-100 μg) of NGF antigen was used toimmunize the HuMab mice intraperitoneally (IP) or subcutaneously (SC).

Immunizations of HuMab transgenic mice were achieved using antigen incomplete Freund's adjuvant and two injections, followed by 2-4 weeks IPimmunization (up to a total of 9 immunizations) with the antigen inincomplete Freund's adjuvant. Several dozen mice were immunized for eachantigen. A total of 118 mice of the HCo7, HCo12, HCo7+HCo12, and KMstrains were immunized with NGF antigen. The immune response wasmonitored by retroorbital bleeds.

To select HuMab mice producing antibodies that bound human NGF, serafrom immunized mice was tested by ELISA as described by Fishwild et alsupra. Briefly, microtiter plates were coated with purified recombinantNGF from E. coli (Example 1) at 1-2 μg/mL in PBS and 50 μL/wellincubated at 4° C. overnight, then blocked with 200 μL/well of 5%chicken serum in PBS/Tween (0.05%). Dilutions of plasma fromNGF-immunized mice were added to each well and incubated for 1-2 hoursat ambient temperature. The plates were washed with PBS/Tween and thenincubated with a goat-anti-human IgG Fc-specific polyclonal reagentconjugated to horseradish peroxidase (HRP) for 1 hour at roomtemperature. Plates were washed with PBS/Tween and incubated with a goatanti-human IgG Fc-specific polyclonal reagent conjugated to horseradishperoxidase (HRP) for 1 hour at room temperature. After washing, theplates were developed with ABTS substrate (Sigma Chemical Co., St.Louis, Mo., Catalog No. A-1888, 0.22 mg/mL) and analyzedspectrophotometrically by determining optical density (OD) atwavelengths from 415-495 nm. Mice with sufficient titers of anti-NGFhuman immunoglobulin were used to produce monoclonal antibodies asdescribed below.

Generation of Hybridomas Producing Human Monoclonal Antibodies to NGF

Mice were prepared for monoclonal antibody production by boosting withantigen intravenously 2 days before sacrifice, and spleens were removedthereafter. The mouse splenocytes were isolated from the HuMab mice andfused with PEG to a mouse myeloma cell line using standard protocols.Typically, 10-20 fusions for each antigen were performed.

Briefly, single cell suspensions of splenic lymphocytes from immunizedmice were fused to one-fourth the number of P3X63-Ag8.653 nonsecretingmouse myeloma cells (ATCC, Accession No. CRL 1580) with 50% PEG (Sigma).Cells were plated at approximately 1×10⁵/well in flat bottom microtiterplates, followed by about a two week incubation in selective mediumcontaining 10% fetal bovine serum, 10% P388D1-(ATCC, Accession No. CRLTIB-63) conditioned medium, 3-5% origen (IGEN) in DMEM (Mediatech,Catalog No. CRL 10013, with high glucose, L-glutamine and sodiumpyruvate) plus 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 mg/mLgentamycin and 1× HAT (Sigma, Catalog No. CRL P-7185). After 1-2 weeks,cells were cultured in medium in which the HAT was replaced with HT.

The resulting hybridomas were screened for the production ofantigen-specific antibodies. Individual wells were screened by ELISA(described above) for human anti-NGF monoclonal IgG antibodies. Onceextensive hybridoma growth occurred, medium was monitored usually after10-14 days. Antibody secreting hybridomas were replated, screened againand, if still positive for human IgG, anti-NGF monoclonal antibodieswere subcloned at least twice by limiting dilution. The stable subcloneswere then cultured in vitro to generate small amounts of antibody intissue culture medium for characterization.

Selection of Human Monoclonal Antibodies that Bind to NGF

An ELISA assay as described above was used to screen for hybridomas thatshowed positive reactivity with NGF immunogen. Hybridomas secreting amonoclonal antibody that bound with high avidity to NGF were subclonedand further characterized. One clone from each hybridoma, which retainedthe reactivity of parent cells (as determined by ELISA), was chosen formaking a 5-10 vial cell bank stored in liquid nitrogen.

An isotype-specific ELISA was performed to determine the isotype of themonoclonal antibodies produced as disclosed herein. In theseexperiments, microtiter plate wells were coated with 50 μL/well of asolution of 1 μg/mL of mouse anti-human kappa light chain in PBS andincubated at 4° C. overnight. After blocking with 5% chicken serum, theplates were reacted with supernatant from each tested monoclonalantibody and a purified isotype control. Plates were incubated atambient temperature for 1-2 hours. The wells were then reacted withvarious human IgG-specific horseradish peroxidase-conjugated goatanti-human polyclonal antisera and plates were developed and analyzed asdescribed above.

Monoclonal antibodies purified from the hybridoma supernatants thatshowed significant binding to NGF as detected by ELISA were furthertested for biological activity using a variety of bioassays as describedbelow.

Example 3

Selecting and Cloning anti-NGF Antibodies with Potent NGF NeutralizingActivity

The effectiveness of the antibodies initially identified in Example 2 asinhibitors of NGF activity (i.e., NGF “neutralization”) was evaluated bymeasuring the ability of each modified peptide to block NGF induction ofvanilloid receptor-1 (VR1) expression.

Dorsal Root Ganglion Neuronal Cultures

Dorsal root ganglia (DRG) were dissected one by one under asepticconditions from all spinal segments of embryonic 19-day old (E19) ratsthat were surgically removed from the uterus of timed-pregnant,terminally anesthetized Sprague-Dawley rats (Charles River, Wilmington,Mass.). DRG were collected in ice-cold L-15 media (GibcoBRL, GrandIsland, N.Y.) containing 5% heat inactivated horse serum (GibcoBRL), andany loose connective tissue and blood vessels were removed. The DRG wererinsed twice in Ca²⁺- and Mg²⁺-free Dulbecco's phosphate buffered saline(DPBS), pH 7.4 (GibcoBRL). The DRG were then dissociated into singlecell suspension using a papain dissociation system (WorthingtonBiochemical Corp., Freehold, N.J.). Briefly, DRG were incubated in adigestion solution containing 20 U/ml of papain in Earle's Balanced SaltSolution (EBSS) at 37° C. for fifty minutes. Cells were dissociated bytrituration through fire-polished Pasteur pipettes in a dissociationmedium consisting of MEM/Ham's F12, 1:1, 1 mg/ml ovomucoid inhibitor and1 mg/ml ovalbumin, and 0.005% deoxyribonuclease I (DNase).

The dissociated cells were pelleted at 200×g for five minutes andre-suspended in EBSS containing 1 mg/ml ovomucoid inhibitor, 1 mg/mlovalbumin and 0.005% DNase. Cell suspension was centrifuged through agradient solution containing 10 mg/ml ovomucoid inhibitor, 10 mg/mlovalbumin at 200×g for six minutes to remove cell debris, and thenfiltered through a 88-μm nylon mesh (Fisher Scientific, Pittsburgh, Pa.)to remove any clumps. Cell number was determined with a hemocytometer,and cells were seeded into poly-ornithine 100 μg/ml (Sigma, St. Louis,Mo.) and mouse laminin 1 μg/ml (GibcoBRL)-coated 96-well plates at10×10³ cells/well in complete medium. The complete medium consisted ofminimal essential medium (MEM) and Ham's F12, 1:1, penicillin (100U/ml), streptomycin (100 μg/ml), and 10% heat inactivated horse serum(GibcoBRL). The cultures were kept at 37° C., 5% CO₂ and 100% humidity.For controlling the growth of non-neuronal cells,5-fluoro-2′-deoxyuridine (75 μM) and uridine (180 μM) were included inthe medium.

Treatment with NGF and Anti-NGF

Two hours after plating, cells were treated with recombinant human β-NGF(Amgen) or recombinant rat β-NGF (R&D Systems, Minneapolis, Minn.) at aconcentration of 10 ng/ml (0.38 nM). Positive controls comprisingserial-diluted anti-NGF antibody (R&D Systems) were applied to eachculture plate. Test antibodies were added at ten concentrations using3.16-fold serial dilutions. All of the samples were diluted in completemedium before being added to the cultures. Incubation time was 40 hoursprior to measurement of VR1 expression.

Measurement of VR1 Expression in DRG Neurons

Cultures were fixed with 4% paraformaldehyde in Hanks' balanced saltsolution for fifteen minutes, blocked with Superblock (Pierce, Rockford,Ill.), and permeabilized with 0.25% Nonidet P-40 (Sigma) in Tris-HCl(Sigma)-buffered saline (TBS) for one hour at room temperature. Cultureswere rinsed once with TBS containing 0.1% Tween 20 (Sigma) and incubatedwith rabbit anti-VR1 IgG for one and one-half hours at room temperature,followed by incubation of Eu-labeled anti-rabbit second antibody (WallacOy, Turku, Finland) for one hour at room temperature. Washes with TBS(3× five minutes with slow shaking) were applied after each antibodyincubation. Enhance solution (150 μl/well, Wallac Oy) was added to thecultures. The fluorescence signal was then measured in a time-resolvedfluorometer (Wallac Oy). VR1 expression in samples treated with themodified peptides was determined by comparing to a standard curve of NGFtitration from 0-1000 ng/ml. Percent inhibition (compared to maximumpossible inhibition) of NGF effect on VR1 expression in DRG neurons wasdetermined by comparing to controls that were not NGF-treated. Resultsare given in Tables 2 and 5.

The cell lines were labeled #110-#129. Antibodies from cell lines #119,#124, and #125 demonstrated extremely potent NGF neutralization activity(FIG. 1). The #124 cell line was a parental cell line, also referred toas 4D4. The #119 and #125 cell lines were subclones of the 4D4 parent.An additional sample from the original vial comprising hybridoma #124(4D4) was grown and labeled #167 (4D4).

Antibodies generated by hybridoma #167 (4D4) were subjected to the sameDRG neuron based NGF neutralization assay as the previous samples.Antibody #167 (4D4) demonstrated strong anti-NGF activity with an IC₅₀of 0.50 nM (FIG. 2), which was consistent with the activity of samples#119, #124, and #125. The activities of the 4 samples are shown in Table2.

TABLE 2 Anti-hNGF activity in DRG cells using 0.38 nM hNGF Code # IC50119 (from 124) <1.2 nM 124 (parent) <0.57 nM 125 (from 124) <0.3 nM 167(from same sample as 124)* 0.50 nM

N-Terminal Sequencing and Mass Spectrometry

Purified anti-NGF hybridoma antibodies samples were prepared for proteinsequencing and LC/MS analysis. Antibodies were purified from conditionedmedia by concentrating the media using Amicon centriprep-30 until thevolume was less than 15 ml. A batch of rProA (Pharmacia) resin waswashed 4× with PBS and a 50% slurry made in PBS following the last wash.An appropriate amount of rProA resin (approximately 5 ug antibody/ulresin but use no less than 50 ul resin) was added to the antibody sampleand incubated overnight at 4° C. The Ab-resin mixture was centrifugedand the unbound fraction was collected. After addition of 0.5 ml PBS andtransfer to a 0.45 um Spin-X (CoStar) tube the sample was centrifuged at10000 rpm for 3 min. The resin was next washed at least 3 times with 0.5ml PBS and then of 0.1M glycine (pH 2.7) was added at 1.5× volume ofresin and incubated for 10 minutes at room temperature followed byanother centrifugation for 3 minutes at 10000 rpm, collecting thesupernatant. This elution step was repeated two more times and then thecombined supernatant was neutralized with 1/25^(th) volume of 1.0 M tris(pH 9.2).

After a final filtering step through a new Spin-x tube (0.2 um) theantibody was quantified using a standard Bradford assay using human IgGas the standard or alternately absorbance at 280 for larger samples. Agel was also run using with 2ug of each sample alongside 2 ug of humanIgG1,k (Sigma). For mass spectrometry, four micrograms of the sampleswere deglycosylated, reduced, and loaded onto an HPLC (HP1090) on-linelinked to a Finingan LCQ mass spectrometer. The light chain wasseparated from the heavy chain by reversed phase HPLC. The light chainsand heavy chains were also collected for N-terminal protein sequencinganalysis.

Both N-terminal sequences of the light chain and heavy chain of thesample of anti-NGF #167 (4D4) antibody matched both N-terminal sequencesof the sample of anti-NGF #119 (4D4) antibody. In addition, the measuredmass of the antibodies indicated that the isolated antibodies from the#167 and #119 hybridomas were the same. The measured, deconvoluted mass(23096) of the light chain of anti-NGF #167 matched the measured mass(23096) of the light chain of anti NGF Ab #119.

Cloning the Anti-NGF Antibody Heavy and Light Chains

The hybridoma expressing the most potent NGF binding monoclonalantibody, 4D4.D7, was used as sources to isolate total RNA using TRIzol®reagent (Invitrogen). First strand cDNA was synthesized using a randomprimer with an extension adapter (5′-GGC CGG ATA GGC CTC CAN NNN NNT-3′)(SEQ ID NO: 33) and a 5′ RACE (rapid amplification of cDNA ends)preparative assay was performed using the GeneRacer™ Kit (Invitrogen)according to instructions from the manufacturer. For preparing completelight chain encoding cDNA, the forward primer was the GeneRacer™ nestedprimer, and the reverse primer was 5′-GGG GTC AGG CTG GAA CTG AGG-3′(SEQ ID NO: 34). For preparing cDNA encoding the variable region of theheavy chain, the forward primer was the GeneRacer™ nested primer and thereverse primer was 5′-TGA GGA CGC TGA CCA CAC G-3′ (SEQ ID NO 35). RACEproducts were cloned into pCR4-TOPO (Invitrogen) and the sequencesdetermined. Consensus sequences were used to design primers forfull-length antibody chain PCR amplification.

For preparing cDNA encoding anti-NGF 4D4.D7 kappa light chain, the 5′PCR primer encoded the amino terminus of the signal sequence, an XbaIrestriction enzyme site, and an optimized Kozak sequence (5′-CAG CAG AAGCTT CTA GAC CAC CAT GGA CAT GAG GGT GCC CGC TCA GCT CCT GGG-3′; SEQ IDNO: 36). The 3′ primer encoded the carboxyl terminus and terminationcodon, as well as a SalI restriction site (5′-CTT GTC GAC TCA ACA CTCTCC CCT GTT GAA GCT C-3′; SEQ ID NO: 37). The resulting PCR productfragment was purified, digested with XbaI and SalI, and then gelisolated and ligated into the mammalian expression vector pDSRα20 (seeInternational Application, Publication No. WO 90/14363, which is hereinincorporated by reference for any purpose. pDSRα20 was produced bychanging nucleotide 2563 in pDSRa19 from a “Guanosine” to an “Adenosine”by site directed mutagenesis.).

For preparing cDNA encoding anti-NGF 4D4.D7 heavy chain the 5′ PCRprimer encoded the amino terminus of the signal sequence, an XbaIrestriction enzyme site, and an optimized Kozak sequence (5′-CAG CAG AAGCTT CTA GAC CAC CAT GGA GTT GGG GCT GTG CTG GGT TTT CCT TGT T-3′; SEQ IDNO: 38). The 3′ primer encoded the carboxyl terminus and terminationcodon, as well as a SalI restriction site (5′-GCA TGT CGA CTC ATT TACCCG GAG ACA GGG AGA G-3′; SEQ ID NO: 39). The resulting product waspurified, digested with XbaI and SalI, gel isolated and ligated into thepDSRα20 vector.

The calculated mass (23099), as determined by translating the nucleotidesequence to predicted amino acids and adding together the molecularweights of the amino acids, of the DNA sequence of the light chain ofanti-NGF Ab 4D4 clone matched the measured mass as determined by massspectrometry. The measured, deconvoluted mass (49479) of the heavy chainof anti-NGF Ab #167 matched the measured mass (49484) of the heavy chainof anti NGF Ab #119 and also matched the theoretical mass (49484) of theDNA sequence of the heavy chain of anti-NGF Ab 4D4 clone (Table 3)within instrumental deviation.

The data of N-terminal protein sequence and LC/MS confirmed thathybridoma #119 expressed the same antibody as hybridoma #167. Inaddition, the calculated mass of the antibodies based on sequencefurther confirmed the observation.

TABLE 3 Summary of Mass Spectrometry Findings Theoretical mass Measuredmass of Measured mass of derived from DNA anti NGF Ab Ab #167 Ab #119sequence of Ab 4D4 light chain 23096 23096 23099 heavy chain 49479 4948449484

Example 4 Expression of Anti-NGF Antibodies in Chinese Hamster Ovary(CHO) Cells

Stable expression of the 4D4 anti-NGF mAb was achieved byco-transfection of 4D4-heavy chain/pDSRα19 IgG2 or 4D4-heavychain/pDSRα19 IgG1 and NGF-kappa/pDSRα19 plasmids into dihydrofolatereductase deficient (DHFR-) serum-free adapted Chinese hamster ovary(CHO) cells using a calcium phosphate method. Transfected cells wereselected in medium containing dialyzed serum but not containinghypoxanthine-thymidine to ensure the growth of cells expressing the DHFRenzyme. Transfected clones were screened using assays such as ELISA inorder to detect the expression of 4D4 anti-NGF mAb in the conditionedmedium. The highest expressing clones were subjected to increasingconcentrations of methotrexate (MTX) for DHFR amplification. MTXamplified clones were screened using assays such as ELISA in order todetect higher expression of 4D4 anti-NGF mAb in the conditioned medium.The highest expressing clones were subjected to subcloning to obtain ahomogeneous population and creation of cell banks.

Recombinant anti-NGF antibodies of the invention can be generated inChinese hamster ovary cells deficient in DHFR using the same protocol asdescribed above for the anti-NGF monoclonal antibody. The DNA sequencesencoding the complete heavy chain or light chain of each anti-NGFantibody of the invention are cloned into expression vectors. CHOd-cellsare co-transfected with an expression vector capable of expressing acomplete heavy chain and an expression vector expressing the completelight chain of the appropriate anti-NGF antibody. For example, togenerate the anti-NGF antibody, cells are co-transfected with a vectorcapable of expressing a complete heavy chain comprising the amino acidsequence as set forth in SEQ ID NO: 40 and a vector capable ofexpressing a complete light chain comprising the amino acid sequence setforth in SEQ ID NO: 44. Table 4 summarizes complete heavy and completelight chains for the 4D4 antibodies having various IgG heavy chainconstant regions.

TABLE 4 Heavy Chain Variable Region + Complete Antibody Heavy ChainConstant Region Heavy Chain 4D4(IgG2) SEQ ID NO: 10 + SEQ ID NO: 4 SEQID NO: 40 4D4(IgG1) SEQ ID NO: 10 + SEQ ID NO: 2 SEQ ID NO: 41 4D4(IgG4)SEQ ID NO: 10 + SEQ ID NO: 6 SEQ ID NO: 42 4D4(IgG3) SEQ ID NO: 10 + SEQID NO: 26 SEQ ID NO: 43 Light Chain Variable Region + Antibody LightChain Constant Region Complete Light Chain 4D4 SEQ ID NO: 12 + SEQ IDNO: 8 SEQ ID NO: 44

Example 5 Characterizing the Activity of Anti-NGF 4D4 Antibodies

Transiently expressed anti-NGF 4D4 antibodies, generated in cells grownunder spinner (S) or roller (R) conditions were tested to confirm theirability to neutralize NGF in a DRG neuron based NGF neutralizationbioassay, performed as described above (Example 3).

The NGF antibodies were expressed transiently in serum-free suspensionadapted 293T cells. Transfections were performed as either 500 mL or 1 Lcultures. Briefly, the cell inoculum (5.0×10⁵ cells/mL X culture volume)was centrifuged at 2,500 RPM for 10 minutes at 4° C. to remove theconditioned medium. The cells were resuspended in serum-free DMEM andcentrifuged again at 2,500 RPM for 10 minutes at 4° C. After aspiratingthe wash solution, the cells were resuspended in growth medium [DMEM/F12(3:1)+1× Insulin-Transferrin-Selenium Supplement+1× Pen Strep Glut+2 mML-Glutamine+20 mM HEPES+0.01% Pluronic F68] in a 1 L or 3 L spinnerflask culture. The spinner flask culture was maintained on magnetic stirplate at 125 RPM which was placed in a humidified incubator maintainedat 37° C. and 5% CO₂. The plasmid DNA was complexed to the transfectionreagent in a 50 mL conical tube. The DNA-transfection reagent complexwas prepared in 5% of the final culture volume in serum-free DMEM. 1 μgplasmid DNA/mL culture was first added to serum-free DMEM, followed by 1μl X-TremeGene RO-1539/mL culture. The complexes were incubated at roomtemperature for approximately 30 minutes and then added to the cells inthe spinner flask. The transfection/expression was performed for 7 days,after which the conditioned medium was harvested by centrifugation at4,000 RPM for 60 minutes at 4° C.

For roller bottle transient transfections, we used 293T adherent cellsgrown and maintained in DMEM supplemented with 5% FBS+1× Non-EssentialAmino Acids+1× Pen Strep Glut+1× Sodium Pyruvate. Approximately, 4-5×10⁷293T cells were seeded in a 850 cm² roller bottles overnight. Thepreviously seeded cells were then transfected the following day usingFuGene6 transfection reagent. The DNA—transfection reagent mixture wasprepared in approximately in 6.75 mL serum-free DMEM. 675 μl FuGene6transfection reagent was first added, followed by 112.5 μg plasmid DNA.The complex was incubated at room temperature for 30 minutes. The entiremixture was then added to a roller bottle. The roller bottle was gassedwith a 5% CO₂ gas mixture, capped tightly and placed in a 37° C.incubator on a roller rack rotating at 0.35 RPM. The transfection wasperformed for 24 hours after which the medium was replaced with 100 mLDMEM+1× Insulin-Transferrin-Selenium Supplement+1× Pen Strep Glu+1×Non-Essential Amino Acids+1× Sodium Pyruvate. Typically, two 100 ml 48hour harvests were obtained from each roller bottle. The harvestedserum-free conditioned medium was pooled together and centrifuged at4,000 RPM for 30 minutes at 4° C.

Both 4D4.IgG1 and 4D4.IgG2 showed potent activity with IC₅₀ values ofabout 0.14 nM to about 0.2 nM against human NGF (FIG. 2). The results ofthe activity assay are summarized in Table 5. The antibodies showedlittle activity against rat NGF (FIG. 3). The results resemble theactivity of the antibodies tested directly from hybridomas describedabove.

TABLE 5 IC50 @ IC50 @ Ab hNGF (nM) rNGF (nM) 4D4.IgG1.R 0.1488 >34 nM4D4.IgG1.S 0.1587 >45 nM 4D4.IgG2.R 0.2047 >59 nM 4D4.IgG2.S 0.2063 >37nM hNGF = human NGF, rNGF = rat NGF, R = Roller culture, S = Spinnerculture

Example 6 Production of Anti-NGF Antibody

Anti-NGF antibody is produced by expression in a clonal line of CHOcells. For each production run, cells from a single vial are thawed intoserum-free cell culture media. The cells are grown initially in aT-flask followed by spinner flasks and then grown in stainless steelreactors of increasing scale up to a 2000 L bioreactor. Production iscarried out in a 2000 L bioreactor using a fed batch culture, in which anutrient feed containing concentrated media components is added tomaintain cell growth and culture viability. Production lasts forapproximately two weeks during which time anti-NGF antibody isconstitutively produced by the cells and secreted into the cell culturemedium.

The production reactor is controlled at a predetermined pH, temperature,and dissolved oxygen level: pH is controlled by carbon dioxide gas andsodium carbonate addition; dissolved oxygen is controlled by air,nitrogen, and oxygen gas flows.

At the end of production, the cell broth is fed into a disk stackcentrifuge and the culture supernatant is separated from the cells. Theconcentrate is further clarified through a depth filter followed by a0.2 μm filter. The clarified conditioned media is then concentrated bytangential flow ultrafiltration. The conditioned media is concentrated15- to 30-fold. The resulting concentrated conditioned medium is theneither processed through purification or frozen for purification at alater date.

Example 7

Cross-Reactivity with other Neurotrophins

The 4D4 antibodies were tested for their cross-reactivity against humanNT3 or human BDNF in different bioassays, including the DRG neuronsurvival assay for human NT3 and the assay of DA uptake in cultured DAneurons for human BDNF.

Treatment of DRG Cultures with NT3, Anti-NT3 and Anti-NGF Antibodies

Two hours after plating, DRG cells (isolation procedure described abovein Example 3) were treated with recombinant hNT-3 100 ng/ml (3.8 nM).Serial-diluted anti-hNT3 antibody (R&D) was used as a positive control.Unknowns (anti-NGF Ab samples) were added at various concentrations with10 point, 3.16 fold serial dilutions. All the samples were diluted incomplete medium before being added to the cultures.

Measurement of MAP2 Expression in DRG Neurons

Cultures were fixed with 4% paraformaldehyde in Hanks' balanced saltsolution for 15 min, blocked with Superblock (Pierce) for 1 hour andpermeabilized with 0.25% Nonidet P-40 (Sigma) in Tris-HCl(Sigma)-buffered saline (TBS) for 1 hour in room temperature (RT).Cultures were rinsed once with TBS containing 0.1% Tween20 (Sigma) andincubated with mouse anti-MAP2 IgG (Chemicon, Temecula, Calif.) for 1.5hour at room temperature, followed by incubation of Eu-labeledanti-mouse secondary antibody (Wallac Oy, Turku, Finland) for 1 hour atroom temperature. Washes with TBS (3×5min with gentle shaking) wereapplied after each antibody incubation. Enhance solution (150 ml/well,Wallac Oy) was added to the cultures and fluorescence signal was thenmeasured in a time-resolved fluorometer (Wallac Oy).

Embryonic Mesencephalic Culture

Embryonic 19 day old (E19) Sprague-Dawley rats (Jackson Labs) were used.Ventral midbrain tissue enriched for dopaminergic neurons was removedand transferred to cold, Dulbecco's phosphate buffered saline (DPBS), pH7.4, without Ca⁺⁺ and Mg⁺⁺ (Gibco). The tissue fragments weredissociated into single cell suspension using a papain dissociationsystem (Worthington Biochemical Corp., Freehold, N.J.). Briefly, tissuefragments were incubated in a digestion solution containing 20 unit/mlpapain in Earle's Balanced Salt Solution (EBSS) at 37° C. for 50 min.Cells were dissociated by trituration through fire-polished Pasteurpipettes in a dissociation medium consisting MEM/Ham's F12 1:1, 1 mg/mlovomucoid inhibitor and 1 mg/ml ovalbumin and 0.005% deoxyribonuclease I(DNase). The dissociated cells were pelleted at 200×g for 5 min andresuspended in EBSS containing 1 mg/ml ovomucoid inhibitor, 1 mg/mlovalbumin and 0.005% DNase. Cell suspension was centrifuged through agradient solution containing 10 mg/ml ovomucoid inhibitor, 10 mg/mlovalbumin at 200×g for 6 min to remove the cell debris; and filteredthrough a 25 μg Nitex nylon mesh (Tetko, Inc.) to remove the clumps. Thedissociated cells were plated in tissue culture plates at a density of100,000/cm². The plates were pre-coated with poly-ornithine 100 μg/ml(Sigma) and mouse laminin 1 μg/ml (Gibco BRL) as previously described(Louis J C et al., J. Pharmacol. Exp. Ther. 1992; 262:1274-1283.). Theculture medium consisted of minimal essential medium (MEM)/Ham's F 12,1:1, 12% horse serum (Gibco), 100 μg/ml transferrin and 2.5 μg/mlinsulin (Sigma). The cultures were kept at 37° C., 5% CO2 and 100%humidity for 6 days.

Treatment of Mesencephalic Cultures with BDNF and Anti-BDNF or Anti-NGF

BDNF at 10 ng/ml was added to the cells 2 hours after plating, followedby serial concentrations of anti-NGF Ab samples. Anti-BDNF antibody(generated at Amgen) was used as a positive control.

DA Uptake in Mesencephalic Neurons

Dopamine uptake assay were carried out as described previously(Friedman, L. and Mytilineou, C., Neuroscience Letters 1987; 79:65-72).At day 6, cultures were washed once with pre-warmed Krebs-Ringer'sphosphate buffer (pH 7.4) containing 5.6 mM glucose, 1.3 mM EDTA and 0.5mM pargylin, a monoamine oxidase inhibitor. The cultures were incubatedin uptake buffer containing 50 nM [³H]DA (NEN) for 60 minutes at 37° C.Uptake was stopped by removing the uptake buffer, and the cultures werewashed three times with Krebs-Ringer's phosphate buffer. Cells werelysed to release [³H]DA by adding a liquid scintillation cocktail,opticphase supermix (Wallac), directly to the cultures. The cell lysateswere then counted for radioactivity in a microbeta-plus liquidscintillation counter (Wallac, Inc.). Low affinity DA uptake wasassessed by adding 0.5 mM GBR12909, a specific inhibitor of the highaffinity DA uptake sites (Heikkila RE and Mazino L, European Journal ofPharmacology 1984; 103:241-8), to the uptake buffer, and subtracted fromthe total uptake amount to obtained the high affinity DA uptake value.

TABLE 6 IC50 @ hNT-3 IC50 @ Antibody (nM) hBDNF (nM) 4D4 (IgG2) >13.75>13.75

Example 8 Identification of an Epitope for Anti-NGF Antibodies EpitopeMapping by Limited Proteolysis

Five micrograms (μg) of NGF were incubated with 4D4 (11 μg) for 30minutes at 4° C. in 01M Tris buffer, pH 7.5. The complex was thendigested with protease (subtilisin) 1 μg at 37° C. for 1 and 2 hours.HPLC peptide maps were compared to each other to find the peptides thatwere protected by the 4D4 antibodies. Limited proteolysis of NGFindicated that several major peptides were initially released from NGF.Of particular interest, peptides S18.3, S18.5, and S34.4 were generatedand protected with antibody from the proteolysis. Other peaks were notsignificantly formed or protected. The protected peptides from twoexperiments (1 hour and 2 hour digestion) are shown in Table 7.

TABLE 7 % protection 1 hour 2 hour digestion digestion S16.1 QAA (96-98)C- — 57 terminal S18.3 FFETK (53-57) Loop 40 45 (SEQ ID region NO: 45)S18.5 SSSHPIFHR N- 40 50 (1-9) terminal (SEQ ID NO: 46) (HWNSY)* (SEQ IDNO: 47) S34.4 NSVEKQYFFETK Loop 69 38 (46-57) region (SEQ ID NO: 48)

The percentage of protection was calculated from the peptide peakheight. S18.5 contained two peptides, but only one peptide (SSSHPIFHR;SEQ ID NO: 46) was protected with the 4D4 antibody, since the otherpeptide peak (HWNSY; SEQ ID NO: 47) was unchanged by the addition of 4D4antibodies, as detected at 280 nm absorbance. Peptide S18.3 was aC-terminal part of S34.4, both from the same loop region. N-terminal andcentral loop regions were also possible epitopes.

Microcon Separation of Digested Peptides

The subtilisin-digested material (3 μg each) was incubated with active4D4 antibodies and an inactive monoclonal antibody (#162) (8 μg) for 30minutes at 4° C. in 0.1 M Tris buffer, pH 7.5. The bound/unboundpeptides were separated by Microcon 10 (Millipore Corp., Bedford, Mass)and both fractions (bound and unbound) were analyzed by HPLC to findpeptides bound to antibodies. Two depleted peaks identified by HPLCcomparison of the unbound fractions after treatment with 4D4 antibodiesand #162 and Microcon separation were recovered, indicating antibodybound peptides. The 4D4 bound peptides were:

(SEQ ID NO: 49) S1 (4.4) ----SRKAVRR (113-119), C-terminal; and (SEQ IDNO: 50) S2 (28.3) ----LYMYL (35-39), Loop region.

An NGF sample was alternatively digested with Lys-C (K) for 24 hours.Cysteine residues were reduced and carboxymethylated without denaturant.The sample was incubated with monoclonal antibodies 4D4 and AMG162,followed by Microcon 100 separation. Bound and unbound fractions wereanalyzed by reversed phase HPLC. Only two peptides were identified asantibody binding K-peptides as indicated below. The calculated mass forthe peptides determined by sequence analysis and the mass spectrometryof the peptides were consistent. The peptides, as indicated below,mapped to the N-terminal and C-terminal region.

(SEQ ID NO: 51) K1 (37.6) ----SSSHPIFHRGEFSVCDSVSVWVGDK

Calculated mass=2821; Observed mass=2828.2; N-terminal

(SEQ ID NO: 52) K2 (39.5) ----QAAWRFIRIDTACVCVLSRK

Calculated mass=2452; Observed mass=2459.5; C-terminal

The preceding epitope mapping experiments indicated that at least threeregions were possible epitopes for the 4D4 antibodies, includingN-terminus (1-9), internal (46-57), and C-terminal (96-98) regions. Inaddition, an AspN digestion revealed that a peptide fragment consistingof -SSHPIFHRGEFSVC- (SEQ ID NO: 53) was protected by the 4D4 antibody,whereas a trypsin digestion showed that a peptide fragment consisting of-SSHPIFHR- (SEQ ID NO: 54) was not protected by the 4D4 antibody. Thus,in the N-terminus, the sequence of GEFSVC (SEQ ID NO: 55) is mostimportant for binding to 4D4 antibodies.

In order to more clearly define the epitope for the anti-NGF antibody4D4.IgG1, a total of 23 peptides were generated synthetically usingstandard techniques based on the entire human mature NGF (hNGF) sequence(Table 8). The peptides were 15 amino acids long, overlapping by 10amino acids, and cysteine-tailed at the C-termini to allow forconjugation to a matrix. The human anti-hNGF Ab 4D4.IgG1 described abovewas used for the mapping experiment.

TABLE 8 SEQ ID Peptide # Sequence NO 33582-27-01 SSSHPIFHRGEFSVC (1-15)56 33582-27-02 IFHRGEFSVADSVSVC (6-20) 57 33582-27-03 EFSVADSVSVWVGDKC(11-25) 58 33582-27-04 DSVSVWVGDKTTATDC (16-30) 59 33582-27-05WVGDKTTATDIKGKEC (21-35) 60 33582-27-06 TTATDIKGKEVMVLGC (26-40) 6133582-27-07 IKGKEVMVLGEVNIN (31-45) 62 33582-27-08 VMVLGEVNINNSVFKC(36-50) 63 33582-27-09 EVNINNSVFKQYFFEC (41-55) 64 33582-27-10NSVFKQYFFETKARDC (46-60) 65 33582-27-11 QYFFETKARDPNPVDC (51-65) 6633582-27-12 TKARDPNPVDSGARDC (56-70) 67 33582-27-13 PNPVDSGARDIDSKHC(61-75) 68 33582-27-14 SGARDIDSKHWNSYC (66-80) 69 33582-27-15IDSKHWNSYATTTHTC (71-85) 70 33582-27-16 WNSYATTTHTFVKALC (76-90) 7133582-27-17 TTTHTFVKALTMDGKC (81-95) 72 33582-27-18 FVKALTMDGKQAAWRC(86-100) 73 33582-27-19 TMDGKQAAWRFIRIDC (91-105) 74 33582-27-20QAAWRFIRIDTAAVC (96-110) 75 33582-27-21 FIRIDTAAVAVLSRKC (101-115) 7633582-27-22 TAAVAVLSRKAVRRAC (106-120) 77 33582-27-23 CAAVAVLSRKAVRRA(107-120) 78

The human NGF peptide fragments were diluted in PBS with 5% DMSO, 1 mMEDTA, pH 6.23. The final peptide concentration was normalized to thesame molar concentration at 55 μM (about 100 μg/ml). Peptides wereincubated in Reacti-Bind Maleimide activated 96 well microtiter plates(Pierce Cat #15150), 100 μl/well, at room temperature for 2 hours andthen at 4° C. overnight with agitation. Human NGF (100 μg/ml) was usedas positive control. The plates were washed with wash buffer (KPL) andblocked with 0.2% non-fat dry milk (in PBS-EDTA buffer, pH 6.23) for 2hours at room temperature and then further blocked with 5% BSA for 1hour. Plates were then incubated with the human anti-NGF antibody atvarious concentrations (0, 3, 10, 30 μg/ml), followed by goat anti-hFcAb-HRP (KPL) for 2 hours. Signal was developed with TMB substrate andread at 450 nm after addition of stop solution (KPL).

Across the 23 human NGF peptides, at least 4 major peaks were observed,indicating 4D4 binding. These peaks corresponded to the followingpeptides: Peptide #1 (SEQ ID NO: 56), SSSHPIFHRGEFSVC (1-15); Peptide#10 (SEQ ID NO: 65), NSVFKQYFFETKARD (46-60); Peptides #16-17 (SEQ IDNO: 71-SEQ ID NO: 72), WNSYATTTHTFVKAL- (76-95); and Peptides #18-21(SEQ ID NO: 73-SEQ ID NO: 76), TTTHT- LSRKC (100-115).

The four binding peaks of 4D4 mapped to the N-terminus, C-terminus,internal domains, as well as loops L2 and L4 in NGF as described inWeismann et al. (1999, Nature 401:184-8). These results are summarizedin Table 9.

TABLE 9 hNGF epitopes N-terminus L2 Internal L4 Internal C-terminusPeptide # peptide # 1 peptide # 10 peptide # 16 peptide # 17 peptide# 19 peptides # 20-21 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: 75- 56), 65), 71), 72), 74), SEQ ID NO: 76),SSSHPI---, NSVFKQ---, WNSYA---, TMDGKQ---, TMDGK---, QAAWR---, 1-1546-60 76-90 81-95 91-105 96-115 Ab binding +++ + ++ ++ +++ ++ signal

Wiesmann et al. solved the crystal structure of hNGF bound to the trkAreceptor, showing that the N-terminus (residues 2-9) was important forreceptor binding (Wiesmann et al., 1999, Nature 401:184-8). The residuesof this segment in NGF are also important for specificity for trkA overtrkB or trkC receptors. Antibody 4D4 is selective for human NGF overmouse/rat NGF, as well as BDNF and NT-3 most likely because N-terminaldifferences between human NGF and other neurotrophins.

Antibody 4D4 binds to peptide #10 (SEQ ID NO: 65) (NSVFK-, 46-60) andpeptide #17 (SEQ ID NO: 72) (TTTHTFVKALTMDGKC, 81-95), correspondingrespectively to loops L2 and L4, which represent two of seven distinctregions with higher than average sequence diversity among theneurotrophins. Swapping experiments between NGF and BDNF of these sevenregions showed that L2 and L4 were important for the biological activityof NGF. Furthermore, substitution of five NT3 residues in loops L2 andL4 with those of NGF introduced NGF-like activity while maintaining NT3activity. Thus, L2 and L4 are likely regions where antibody 4D4 bindselectively to NGF rather than to BDNF or NT-3.

Antibody 4D4 also binds to peptide #16 (SEQ ID NO: 71) (WNSYATTTHTFVKAL,76-90), matching an internal domain of the NGF crystal structure. Thisregion is 100% homologous between human NGF and mouse NGF, but distinctfrom other neurotrophins. 4D4 showed much weaker activity againstrat/mouse NGF when compared to its activity against human NGF. Thus,binding to this part of NGF is most likely not critical for speciesspecificity but is important for selectivity amongst neurotrophins.

Antibody 4D4 also binds to the C-terminal region of NGF (peptides #19-21(SEQ ID NO: 74-SEQ ID NO: 76)TMDGK-LSRKC, 91-115), which is one of theregions of human NGF that distinguishes NGF from other neurotrophins(BDNF and NT3). Binding to this region helps to explain why 4D4 is notactive against other neurotrophins. Furthermore, there is a single aminoacid difference between human NGF and mouse NGF in the C-terminus,suggesting that this single amino acid may be one of the reasons 4D4 isselective for human NGF over rat/mouse NGF, similar to the N-terminuswhere species differences are observed.

Lastly, 4D4 also interacts with an internal domain described by peptide#10 (SEQ ID NO: 65) (-KARDC, 50-60) of human NGF, which is an importantregion for NGF binding preferentially to trkA, rather than trkB or trkC,further explaining its selective neutralization activity against humanNGF.

Example 9 Affinity Measurement of Monoclonal Antibodies by KinExA

Binding of Ab 4D4 (38859-80) to huNGF (29714-91) was tested on KinExA.Briefly, Reacti-Gel 6× (Pierce) were pre-coated with huNGF and blockedwith BSA. 10 pM and 30 pM of Ab 4D4 samples were incubated with variousconcentrations of huNGF (Amgen) at room temperature for 8 hours beforerun through the huNGF-coated beads. The amount of the bead-boundantibody was quantified by fluorescent (Cy5) labeled goat anti-human-IgGantibody (Jackson Immuno Research). The binding signal was proportionalto the concentration of free antibody at equilibrium. Dissociationequilibrium constant (K_(D)) was obtained from nonlinear regression ofthe competition curves using a dual-curve one-site homogeneous bindingmodel (KinEX™ software). The K_(D) was about 4 pM for Ab 4D4 binding tohuNGF.

Example 10

Identification of additional Anti-NGF Antibodies

Additional anti-NGF antibodies (designated 14D10, 6G9, 7H2, 14F11, and4G6), generated and identified as described in Examples 2 and 3 above,were selected for further study. Briefly, conditioned media was testedfor binding activity. Antibodies from the media were purified andsequenced. The predicted mass was compared with mass spectrometry dataof antibodies from the conditioned media. The antibodies were cloned.Two of the clones were expressed in CHO cells and tested for activity asdescribed above. The results are shown in Table 10.

TABLE 10 IC50 @ IC50 @ IC50 @ IC50 @ hNGF rNGF Molecular hNGF rNGF clone(nM) (nM) Notes Clone (nM) (nM) 7H2 3.294 1.748 cloned 7H2-rFc 0.9630.792 6H9 3.172 1.699 cloned 6H9-rFc 13.93 0.653 14D10 0.3918 >13 cloned14D11 0.2803 >20 cloned 4G6 0.414 >10 cloned

The sequences of the light and heavy chain variable regions of theseantibodies were then compared to the 4D4 antibody sequence, as well asto each other (FIGS. 5 and 6). The percent homologies of the heavy chainvariable regions as identified from these comparisons are shown in Table11. The percent homologies of the light chain variable regions are shownin Table 12. In addition, the percent homologies of the CDR regions ofthe various antibodies are shown in FIGS. 5-10.

TABLE 11 4D4 VH 14D10 VH 6H9 VH 7H2 VH 14D11 VH 4G6 VH 4D4 VH 100% 70.9%70.1% 75.6% 47.2.%  73.4% 14D10 VH  100% 95.3%   85% 54.3% 81.1% 6H9 VH 100% 86.6% 54.3% 81.1% 7H2 VH  100% 51.2% 79.8% 14D11 VH  100% 56.8%4G6 VH  100%

TABLE 12 V4D4 14D11 4G6a 4G6b 4G6c 14D10 4G6d VK LC LC LC LC LC 6H9 LCLC 7H2 LC 4G6e V4D4 100% 89% 91% 72% 74% 69% 71% 71% 70% 73% VK 14D11100% 94% 68% 71% 67% 68% 68% 68% 70% LC 4G6a 100% 69% 74% 68% 70% 70%69% 71% LC 4G6b 100% 87% 83% 86% 86% 86% 96% LC 4G6c 100% 91% 94% 94%94% 91% LC 14D10 100% 91% 94% 94% 86% LC 6H9 100% 99% 98% 89% LC 4G6d100% 99% 89% LC 7H2 100% LC 4G6e 100%

Example 11 Safety and Tolerability of Subcutaneous NGF Antibody inHumans

The objectives of this study were to evaluate the safety andtolerability of multiple subcutaneous (SC) doses of antibodies to nervegrowth factor (NGF antibody) in subjects with osteoarthritis (OA) kneepain and to examine the serum pharmacokinetics (PK) of NGF antibodiesfollowing administration of multiple SC doses of the NGF antibodies tosubjects with OA knee pain. Clinical benefits of multiple SC doses wereassessed by the Western Ontario and McMaster Universities ArthritisIndex [WOMAC™3.1].

A phase I, sequential randomized, double-blind placebo-controlled,multiple dose, dose escalation study in subjects with OA knee pain wasconducted. The study design included 4 cohorts of subjects with 8subjects in each cohort (n=32). Three dose levels (3 mg, 10 mg, 20 mg)of NGF antibodies were used. The NGF antibodies used in this study werefully human monoclonal antibodies. To produce the NGF antibodies, cellsfrom master cell bank vials were serially expanded in suspension overapproximately 3-4 weeks. Cells were thawed into shaker flasks, followedby spinner flasks, a series of seed bioreactors, and finally into alarge bioreactor for production. Production lasts approximately 2 weeks,during which NGF antibodies are constitutively produced by the cells andsecreted into the cell culture medium. The NGF antibody recovery processremoves cellular debris through a combination of centrifugation, depthfiltration and membrane filtration followed by initial purification withProtein A affinity chromatography and low pH viral inactivation. Afterrecovery, NGF antibodies were purified to drug substance using thefollowing unit operations: anion-exchange (AEX) chromatography, cationexchange (CEX) chromatography, viral filtration, andultrafiltration/dialfiltration. NGF antibody drug product wasmanufactured using filtration of drug substance followed by sterilefiltration and vial filling.

Although variability of the efficacy data was large and the study wasnot powered to evaluate the statistical significance of the efficacymeasures, pain-related treatment effects seemed apparent. Mean totalWOMAC scores, mean WOMAC subcategory scores, mean patient global diseaseassessment, and mean physician global disease assessments showed ageneral trend of improvement at the first time point after the firstdosing in the treatment groups with effects maintained throughout thedosing period. These trends were not as apparent in the placebo group.After the final dose of the NGF antibody, treatment effects trendedtowards baseline over time.

After single or multiple SC dose administration to subjects with OA kneepain, NGF antibody exposure appeared to increase approximately inproportion to the dose in the dose range of 3 mg to 20 mg. MedianT_(max) ranged from 7.5 to 11.5 days. Adverse events (AEs) were reportedas treatment-related for 10 subjects (56%) in the treatment groups andfor 2 subjects (33%) in the placebo group. No subjects discontinued thestudy early because of AEs. However, because of Grade 2 neurosensoryAEs, the protocol-specified stopping rule became applicable such that 5subjects in a cohort (2 placebo, 3 treated) did not receive the fourthand final dose of the investigational product. Neurosensory AEs appearedto be dose-dependent.

In male and female OA subjects between the ages of 36 and 63 years ofage, multiple SC dosing appeared to be well tolerated at the dosesadministered in this study except for treatment-emergent, possibledose-dependent neurosensory events (mild or moderate in severity)reported for 4 of 6 subjects who received 20 mg of the antibody. NGFantibody exposure appeared to increase approximately in proportion tothe dose in the dose range of 3 mg to 20 mg. Terminal phase half-life(t_(1/2,z)) ranged from 20.3 to 26.4 days.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

1. A method of treating a condition caused by increased expression ofnerve growth factor (NGF) or increased sensitivity to NGF comprisingadministering to a patient orally, through injection by intravenous,intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, intralesional or subcutaneous routes, by sustained releasesystems or by implantation devices a pharmaceutically effective amountof an NGF antibody.
 2. The method of claim 1, wherein the injection is asubcutaneous injection.
 3. The method of claim 2, wherein thepharmaceutically effective amount of the NGF antibody is from about 3 mgto about 30 mg per subcutaneous injection.
 4. The method of claim 2,wherein the subcutaneous injection comprises multiple subcutaneousinjections.
 5. The method of claim 1, wherein the condition is acutepain, dental pain, pain from trauma, surgical pain, pain resulting fromamputation or abscess, causalgia, demyelinating diseases, trigeminalneuralgia, cancer, chronic alcoholism, stroke, thalamic pain syndrome,diabetes, acquired immune deficiency syndrome (“AIDS”), toxins,chemotherapy, general headache, migraine, cluster headache,mixed-vascular or non-vascular syndromes, tension headache, generalinflammation, arthritis, rheumatic diseases, lupus, osteoarthritis,fibromyalgia, inflammatory bowel disorders, irritable bowel syndrome,inflammatory eye disorders, inflammatory or unstable bladder disorders,psoriasis, skin complaints with inflammatory components, sunburn,carditis, dermatitis, myositis, neuritis, collagen vascular diseases,chronic inflammatory conditions, inflammatory pain and associatedhyperalgesia and allodynia, neuropathic pain and associated hyperalgesiaor allodynia, diabetic neuropathy pain, sympathetically maintained pain,deafferentation syndromes, asthma, epithelial tissue damage ordysfunction, herpes simplex, disturbances of visceral motility atrespiratory, genitourinary, gastrointestinal or vascular regions,wounds, burns, allergic skin reactions, pruritis, vitiligo, generalgastrointestinal disorders, colitis, gastric ulceration, duodenalulcers, vasomotor or allergic rhinitis, or bronchial disorders,dysmenorrhoea, dyspepsia, gastroesophageal reflux, pancreatitis, orvisceralgia.
 6. The method of claim 5, wherein the osteoarthritis isosteoarthritis knee pain.
 7. The method of claim 6, wherein theinjection is a subcutaneous injection.
 8. The method of claim 7, whereinthe pharmaceutically effective amount of the NGF antibody is from about3 mg to about 30 mg per subcutaneous injection.
 9. The method of claim1, comprising administering a pharmaceutical composition comprising apharmaceutically acceptable carrier and an isolated antibody, whereinthe antibody comprises a light chain comprising SEQ ID NO. 44 and aheavy chain comprising SEQ ID. NO.
 40. 10. The method of claim 9,wherein the heavy chain and light chain of the antibody are connected bya flexible linker to form a single chain antibody.
 11. The method ofclaim 1, wherein the NGF antibody is a Fab′ antibody.
 12. The method ofclaim 1, wherein the NGF antibody is a (Fab′)₂ antibody.
 13. The methodof claim 1, wherein the NGF antibody is fully human.
 14. The method ofclaim 1, wherein the NGF antibody is humanized.
 15. The method of claim1, wherein the NGF antibody inhibits NGF signaling.
 16. The method ofclaim 1, wherein the NGF antibody comprises a light chain comprising SEQID NO: 44 and a heavy chain comprising SEQ ID NO: 40 and the heavy chainand light chain of the antibody are connected by a flexible linker toform a single chain antibody.
 17. The method of claim 16, wherein theNGF antibody is a single-chain Fv antibody.
 18. The method of claim 16,wherein the NGF antibody is a Fab′ antibody.
 19. The method of claim 16,wherein the NGF antibody is a (Fab′)₂ antibody.
 20. The method of claim16, wherein the NGF antibody is fully human.
 21. The method of claim 16,wherein the NGF antibody is humanized.
 22. The method of claim 16,wherein the NGF antibody inhibits NGF signaling.
 23. The method of claim16, wherein the NGF antibody dissociates from a human NGF polypeptidewith a K_(D) of about 1×10⁻⁹ or less and neutralizes human NGFbioactivity in a standard in vitro assay with an IC₅₀ of about 1×10⁻⁸ orless.
 24. The method of claim 16, wherein the NGF antibody dissociatesfrom a human NGF polypeptide with a K_(D) of about 1×10⁻¹⁰ or less andneutralizes human NGF bioactivity in a standard in vitro assay with anIC₅₀ of about 1×10⁻⁹ or less.
 25. The method of claim 16, wherein theNGF antibody dissociates from a human NGF polypeptide with a K_(D) ofabout 1×10⁻¹¹ or less and neutralizes human NGF bioactivity in astandard in vitro assay with an IC₅₀ of about 0.2×10⁻⁹ or less.